Fully human monoclonal antibodies to IL-13

ABSTRACT

The present invention is related to antibodies directed to IL-13 and uses of such antibodies. For example, in accordance with the present invention, there are provided human monoclonal antibodies directed to IL-13. Isolated polynucleotide sequences encoding, and amino acid sequences comprising, heavy and light chain immunoglobulin molecules, particularly sequences corresponding to contiguous heavy and light chain sequences spanning the framework regions (FR&#39;s) and/or complementarity determining regions (CDR&#39;s), are provided. Additionally, methods of using these antibodies to treat patients are also provided. Additionally, IL-13 dependent biomarkers and methods of their identification and use are also provided.

RELATED APPLICATIONS

This Application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application Nos. 60/629,135 and 60/728,604, filed Nov. 17,2004, and Oct. 19, 2005, respectively, which are incorporated byreference in their entireties.

FIELD OF INVENTION

The present invention relates generally to compounds that relate toIL-13. More specifically, the compounds can bind to interleukin-13. Morespecifically, the invention relates to human monoclonal antibodies thatspecifically bind interleukin-13 and can affect IL-13 activity.

BACKGROUND OF THE INVENTION

Interleukin-13 (IL-13) is a cytokine that was first recognized for itseffects on B cells and monocytes, where it up-regulates class IIexpression, promotes IgE class switching and inhibits inflammatorycytokine production. The IL-13 receptor shares the IL-4 receptor alphachain with the IL-4 receptor. As a result, IL-13 has many similarbiological activities to IL-4.

IL-13 inhibits proinflammatory cytokine release and has ananti-inflammatory activity in vivo. IL-13 plays a role in IgE mediatedallergic responses and is the central mediator of allergic asthma(Wills-Karp M., Curr. Opin. Pulm. Med., 2003; 9:21-27). In the lung itregulates eosinophilic inflammation, mucus secretion, and airwayhyperresponsiveness. In addition to asthma, IL-13 is implicated in thepathogenesis of a large number of diseases (Wynn T A. Annu. Rev.Immunol. 2003. 21:425-456).

SUMMARY OF THE INVENTION

Some aspects of the invention relate to an isolated human antibody thatbinds to IL-13. In some embodiments, the isolated human antibody bindsto human IL-13 with a K_(D) of less than 170 pM. In some embodiments,the isolated human antibody binds to IL-13 with a K_(D) of less than 50pM. In some embodiments, the antibody can inhibit airwayhyperresponsiveness. In some embodiments, the antibody allows for acomplete reversal of airway hyperresponsiveness. In some embodiments,the antibody can reduce mucus production in the lung. In someembodiments, the antibody allows for the reduction of at least about 30%of the mucus production. In some embodiments, the antibody can inhibitan IL-13 related disorder selected from the group consisting of: chronicobstructive pulmonary disease, chronic bronchitis, emphysema, asthma. Insome embodiments, the antibody binds to an epitope of IL-13 thatprevents IL-13 from signaling through an interaction with an alpha 1IL-13 receptor. In some embodiments, the antibody has an IC₅₀ of no morethan 60 pM in an eotaxin release assay with 300 pM of IL-13. In someembodiments, the antibody binds to human IL-13 but does not detectablybind to murine IL-13.

Some aspects of the invention relate to a method of treating an IL-13related disorder. In some embodiments, the method comprisesadministering an effective amount of a human antibody that binds toIL-13 to a subject in need of treatment, wherein the isolated humanantibody binds to IL-13 with a K_(D) of no more than 170 pM, therebytreating the IL-13 related disorder.

In some embodiments, the treatment of airway hyperresponsiveness, mucusproduction, or both in a subject occurs as a prophylactic treatment, andthe method further comprises the step of identifying a patient at riskof developing airway hyperresponsiveness, mucus production or both. Insome embodiments, the IL-13 related disorder is selected from the groupconsisting of: airway hyperresponsiveness, mucus production, asthma orsome combination thereof. In some embodiments, the antibody has a K_(D)of no more than about 10 pM. In some embodiments, the IL-13 relateddisorder is hodgkins lymphoma. In some embodiments, the effective amountis an amount that is sufficient to lower an amount of a detectablebiomarker in a patient. In some embodiments, an effective amount is anamount that can reduce the amount of IL-13 present in a subject by andsignificant amount, for example 1-10, 10-20, 20-30, 30-40, 40-50, 50-60,60-70, 70-80, 80-90, 90-95, 95-99, 99-100%. In some embodiments, thebiomarker is selected from the group consisting of: C10, TARC, eotaxin,and some combination thereof. In some embodiments, the effective amountis at least an amount sufficient to inhibit at least some cellproliferation of HDLM-2, L-1236 cells, or some combination thereof. Insome embodiments, the IL-13 related disorder relates to the expressionof CD23.

Other aspects of the invention relate to an isolated human antibody thatbinds to IL-13, wherein the isolated human antibody binds to IL-13 in amanner such that IL-13 can still bind to IL-13 receptor alpha 2, andwherein the isolated human antibody binds to IL-13 in a manner so as toprevent IL-13 from binding to IL-13 receptor alpha 1.

Other aspects of the invention relate to a method for measuring aninhibition of IL-13 activity. In some embodiments, the method comprisesadministering an antibody to a sample or a subject and measuring anamount of a biomarker released, wherein a decrease in the amountbiomarker released correlates with an inhibition of IL-13 activity.

Other aspects of the invention relate to an isolated human antibody toIL-13. The antibody binds IL-13 and IL-13Q110R with a K_(D) that is lessthan 170 pM and binds to both IL-13 and IL-13Q110R with K_(D)s that arewithin 50% of each other. In some embodiments, the antibody binds toIL-13 and IL-13Q110R with effectively the same K_(D).

Other aspects of the invention relate to a kit for treating IL-13related disorders. In some embodiments, the kit comprises an IL-13antibody and a biomarker detector for detecting biomarker levels. Insome embodiments, the biomarker is selected from the group consistingof: eotaxin, TARC, C10, and some combination thereof. In someembodiments, the biomarker detector comprises an antibody to a proteinselected from the group consisting of: eotaxin, TARC, C10, or somecombination thereof.

Other aspects of the invention relate to a method of treating an IL-13related disorder. In some embodiments, the method comprisesadministering a first amount of a human antibody that binds to IL-13 toa subject in need of treatment, wherein the isolated human antibodybinds to IL-13 with a K_(D) of no more than 100 pM, detecting an amountof a biomarker to determine a level of IL-13 related activity occurringafter the administration of the first amount of the human antibody, anddetermining if more or less treatment is required based upon the amountof IL-13 activity indicated by said detection of the biomarker. In someembodiments, the method further comprises administering a second amountof a human antibody, wherein said second amount of the antibody is basedupon the amount of the biomarker detected. In some embodiments, thesecond amount of a human antibody is of an antibody that is differentfrom the amount of antibody administered in the first amount of thehuman antibody. In some embodiments, the determination is achieved bycomparing the amount of biomarker determined to either a standard amountof the biomarker for a healthy subject or a set goal amount of thebiomarker for a subject. In some embodiments, the biomarker is selectedfrom the group consisting of: TARC, eotaxin, C10, and some combinationthereof. In some embodiments, the subject in need of treatment is asubject that will benefit from a prophylactic treatment for theprevention of IL-13 related disorders. In some embodiments, the subjectin need of treatment is a subject that will benefit from a therapeutictreatment regarding IL-13 related disorders.

Other aspects of the invention relate to a method for treating asthma.In some embodiments, the method comprises identifying a subject withasthma and administering an effective amount of a human antibody thatbinds to human IL-13 with a K_(D) of no more than about 170 pM. In someembodiments, the effective amount is determined by monitoring a level ofa biomarker, wherein said effective amount is achieved once the level ofthe biomarker decreases. In some embodiments, the biomarker is selectedfrom the group consisting of: eotaxin, C10, TARC, and some combinationthereof, wherein an increase or lack of sufficient decrease in the levelof the biomarker indicates that additional antibody should beadministered. In some embodiments, the patient with asthma is identifiedby the subject having a higher level of a biomarker than a controlgroup.

Other aspects of the invention relate to a method of treating a symptomof an IL-13 related disorder. In some embodiments, the method comprisesidentifying a subject having a symptom of an IL-13 related disorder byidentifying a subject with a symptom that is common to asthma andadministering an effective amount of a human antibody to IL-13 to saidsubject, wherein said effective amount is sufficient to reduce saidsymptom. In some embodiments, the symptom is selected from the groupconsisting of the following: airwayhyperresponsiveness, excess mucusproduction, leukocyte recruitment in bronchoalveolar lavage fluid(BALF), and any combination thereof. In some embodiments, the symptom isa symptom that results in a subject when significant amounts of IL-13are administered to the subject.

Other aspects of the invention relate to the use of an effective amountof a human antibody that binds to IL-13 in the preparation of amedicament for treating an IL-13 related disorder, wherein the isolatedhuman antibody binds to IL-13 with a K_(D) of no more than 170 pM. Insome embodiments, the IL-13 related disorder is treatment of airwayhyperresponsiveness, mucus production, or both, and wherein thetreatment is a prophylactic treatment. In some embodiments, the IL-13related disorder is selected from the group consisting of: airwayhyperresponsiveness, mucus production, asthma or some combinationthereof. In some embodiments, the antibody has a K_(D) of no more thanabout 10 pM. In some embodiments, the IL-13 related disorder is hodgkinslymphoma. In some embodiments, the effective amount is an amount that issufficient to lower an amount of a detectable biomarker in a patient. Insome embodiments, the effective amount is an amount that is sufficientto lower the amount of IL-13 in a subject by any significant amount, forexample, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-95, 95-99, 99-100 percent. In some embodiments, the medicamentfurther comprises a biomarker detector that can report on a level of abiomarker. In some embodiments, the biomarker is selected from the groupconsisting of: C10, TARC, eotaxin, and some combination thereof. In someembodiments, the biomarker detector comprises an antibody that binds tothe biomarker. In some embodiments, the effective amount is at least anamount sufficient to inhibit at least some cell proliferation of HDLM-2,L-1236 cells, or some combination thereof. In some embodiments, theIL-13 related disorder relates to the expression of CD23.

Other aspects of the invention relate to the use of a biomarker detectorin the production of a medicament for monitoring the level of abiomarker as it reports on an IL-13 related disorder.

Other aspects of the invention relate to the use of an antibody to IL-13in the production of a medicament, wherein the antibody binds to IL-13in a manner such that IL-13 can still bind to IL-13 receptor alpha 2,thereby allowing IL-13 receptor alpha 2 to still function as a sink forIL-13, thereby enhancing the clearance of IL-13 from a subject.

Some embodiments of the invention relate to isolated monoclonalantibodies, or fragments thereof, that specifically bind to IL-13. Itwill be appreciated that in these embodiments, the isolated antibodiescan be monoclonal antibodies, chimeric antibodies and/or human orhumanized antibodies. Preferably, the antibodies are human or fullyhuman monoclonal antibodies and bind to IL-13 with an equilibriumdissociation constant lower than 200 pM. In one embodiment, theantibodies bind to IL-13 with a dissociation constant lower than 100 pM.In another embodiment, the antibodies bind to IL-13 with an equilibriumdissociation constant lower than 55 pM. In some embodiments, theantibodies binds to IL-13 with a K_(D) lower than 200 pM or even lowerthan 50 pM. In some embodiments, these antibodies, when administered toa patient, inhibit partially or completely airway hyperresponsiveness.

In one embodiment, the antibody is the “623” antibody discussed belowhaving heavy chain SEQ ID NO: 50 and light chain SEQ ID NO: 52. Inanother embodiment, the antibody is the “731” antibody having heavychain SEQ ID NO: 38 and light chain SEQ ID NO: 40.

In another embodiment of the invention, the antibodies preferably bindto specific epitopes of IL-13. In one embodiment, the antibody binds toan epitope including amino acids 21-33 of IL-13. In another embodiment,the antibody binds to an epitope including amino acids 109-121 of IL-13.In yet another embodiment, the antibody binds to an epitope includingamino acids 111-128. Still another embodiment is an antibody that bindsto an epitope including amino acids 45-108 of IL-13. Other embodimentsinclude antibodies that bind to amino acids 70-80 or 83-92 of IL-13.Still another embodiment is an antibody that binds to a specific Helixof IL-13. For example, antibodies that bind to HelixA, HelixC or HelixDof IL-13 are within the scope of the invention. In another embodiment,the antibody binds to an epitope on IL-13, wherein the epitope includesamino acids 20 through 29 of IL-13.

Another embodiment of the invention is an antibody that has a specificheavy chain amino acid sequence. For example, one embodiment is anantibody that specifically binds to IL-13 and has a heavy chain aminoacid sequence shown in Table 18 below. Preferably, such antibodies alsohave a light chain amino acid sequence as shown in Table 19 or 20 below.In one embodiment, the antibodies include human heavy chainimmunoglobulin molecules represented by SEQ ID NOs: 2, 6, 10, 14, 18,22, 26, 30, 34, 38, 42, 46, 50, 54, 58, and 81-88, for example, and thehuman kappa light chain immunoglobulin molecules represented by SEQ IDNOs 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, and 89-94,for example. Other embodiments include antibody molecules formed bycombinations comprising the heavy chain immunoglobulin molecules withlight chain immunoglobulin molecules, such as the kappa light chainimmunoglobulin molecules, and vice versa, as well as fragments andanalogs thereof. In some embodiments, the antibody has a sequence fromthe heavy chain CDR1, CDR2, CDR3, FR1, FR2, FR3, and/or FR4 or any ofthe sequences listed in Table 18. In some embodiments, the antibody hasa sequence from the light chain CDR1, CDR2, CDR3, FR1, FR2, FR3, and/orJ or any of the sequences listed in Tables 19 and 20.

In some embodiments, the antibody above, when administered to a patient,can reduce mucus production in the lung. The reduction can be a partial,or a complete reduction. In some embodiments, these antibodies can beeffective for reducing mucus production in a mouse. In some embodiments,the antibody, when administered to a human patient, can inhibit chronicobstructive pulmonary disease, chronic bronchitis, emphysema, and/orasthma.

In some embodiments, the antibody binds to a region of IL-13 thatprevents IL-13 from signaling through an interaction with an IL-13receptor. In some embodiments, the antibody can allow binding of IL-13to an IL-13 receptor alpha 2, while inhibiting IL-13 signaling throughthe interaction with the IL-13 receptor alpha 1. In some embodiments,the isolated fully human antibody can inhibit IL-13 dependent signalingby blocking IL-13 from binding to the IL-13 receptor alpha 1.

In some embodiments the antibody has a K_(D) of no more than 3 nM for amacaque IL-13 protein.

Another embodiment of the invention is a method of inhibiting airwayhyperresponsiveness. The method comprises administering an effectiveamount of a fully human antibody that binds to IL-13 to a subject inneed of treatment. In one embodiment, the isolated fully human antibodybinds to IL-13 with a K_(D) of no more than 100 pM and thereby inhibitsairway hyperresponsiveness.

Another embodiment of the invention is a method of inhibiting mucusproduction. The method comprises administering an effective amount of afully human antibody that binds to IL-13 to a subject in need of suchtreatment. Preferably, the isolated fully human antibody binds to IL-13with a K_(D) of no more than 100 pM and thereby inhibits mucusproduction.

In some embodiments, the antibody binds to IL-13 with a K_(D) of no morethan about 50 pM. In another embodiment, the antibody binds to IL-13with a K_(D) of no more than about 10 pM. In some embodiments, themethod is performed on a mouse, while in other embodiments, the methodis performed on a human.

In some embodiments, the above antibody has an IC₅₀ of no more thanabout 100 pM, about 50 pM, about 30 pM, and/or about 20 pM.

Still another embodiment is a method of enhancing the clearance of IL-13from a subject. In some embodiments, the method comprises administeringan antibody that can bind to IL-13 to a subject. The antibody preferablybinds to IL-13 in a manner such that IL-13 can still bind to IL-13receptor alpha 2 and thereby allows IL-13 receptor alpha 2 to stillfunction as a “sink” for IL-13, thereby enhancing the clearance of IL-13from a subject. One such example of an antibody that may be capable ofdoing this is mAb 731. In another embodiment, the antibody binds toIL-13 in a manner such that IL-13 cannot bind to either of the alpha 1or alpha 2 receptors.

One other embodiment is a method of suppressing the level of IL-13dependent activity in a subject. In some embodiments, the methodcomprises administering an antibody to IL-13 to a subject. The antibodybinds to the IL-13 in a manner so as to prevent the IL-13 from signalingthrough its endogenous receptor. Preferably, the signaling requires theIL-13 to bind to an IL-13 receptor alpha 1, and the antibody binding tothe IL-13 does not significantly interfere with the IL-13 binding to anIL-13 receptor alpha 2. In some embodiments, the method furthercomprises monitoring a level of an IL-13 dependent biomarker andadjusting the amount of antibody administered accordingly. In someembodiments, the antibody is mAb 623, 731, and an antibody that bind tothe same epitope as the 623 or 731 antibody. In some embodiments thebiomarker is eotaxin, C10 (a CC chemokine), and/or thymus- andactivation-regulated chemokine (TARC). Additionally contemplated arebiomarker detectors, which are compositions, such as proteins (e.g.,antibodies) that can be employed in some manner to detect the level of abiomarker in a sample.

In some embodiments, the method allows for measuring the inhibition ofIL-13. The method comprises applying a candidate antibody to a samplecomprising IL-13 and measuring the amount of eotaxin released. Theinhibition of eotaxin correlates with the binding of an antibody toIL-13. In some embodiments, the method comprises measuring the amount ofC10 and/or TARC. In some embodiments, the method can be used to identifya subject suffering from an IL-13 related disorder.

In some embodiments, an isolated variant of IL-13 comprising a pointmutation at amino acid position 110 is provided. The point mutationresults in a change from a glutamine at position 110 to an arginineresidue.

Some embodiments of the invention include an isolated fully humanantibody to IL-13, wherein the antibody specifically binds to a variantof IL-13. In some embodiments, the antibody may specifically bind to theIL-13 variant IL-13Q110R more strongly than the antibody binds to IL-13.In some embodiments, the antibody may bind specifically to the variantIL-13Q110R, but does not bind to the wild-type IL-13. In someembodiments the antibody binds to the variant with a K_(D) of no morethan 100 pM. In some embodiments, the antibody may bind to IL-13 morestrongly than it does the IL-13 variant IL-13Q110. In some embodiments,the antibody may bind specifically to the wild-type IL-13, but does notdetectably bind to the variant IL-13Q110R. In some embodiments, theantibody binds to IL-13 as effectively as it binds to IL-13Q110R. Insome embodiments, there is less than a 20% difference in the K_(D) ofthe fully human mAb for IL-13 and IL-13Q110R, for example, less than20-15, 15-10, 10-8, 8-6, 6-4, 4-2, 2-1, 1-0 percent difference in theK_(D)s of the antibody.

In some embodiments, a transgenic mouse is provided. The mouse ishumanized and expresses human IL-13. In some embodiments, the mouse issusceptible to allergen-induced airway hyper-reactivity.

In some embodiments, the above monoclonal antibody or antigen-bindingportion thereof of is a monoclonal antibody.

In some embodiments, a composition comprising the above monoclonalantibody or antigen-binding portion and a pharmaceutically acceptablecarrier is provided.

In some embodiments a kit for treating IL-13 related disorders containsan IL-13 antibody. In some embodiments, the kit comprises an IL-13antibody disclosed herein and instructions for administering the IL-13antibody to a subject. In some embodiments, the kit further includes anIL-13 dependent biomarker for determining if more or less antibody isrequired for treating the IL-13 related disorder.

It will also be appreciated that embodiments of the invention are notlimited to any particular form of an antibody. For example, theantibodies provided may be a full length antibody (e.g. having an intacthuman Fc region) or an antibody fragment (e.g. a Fab, Fab′ or F(ab′)₂).In addition, the antibodies may be manufactured from a hybridoma thatsecretes the antibody, or, recombinantly, from a cell that has beentransformed or transfected with a gene or genes encoding the antibody.

Other embodiments include isolated nucleic acid molecules encoding anyof the antibodies described herein.

In yet further embodiments, the invention provides an isolatedpolynucleotide molecule described herein. As will be appreciated by oneof skill in the art, in some embodiments, any of the presently disclosedantibodies or variants thereof can be used in or for any of thedescribed embodiments or aspects, as appropriate.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be better understood from the Detailed Descriptionand from the appended drawings, which are meant to illustrate and not tolimit the invention.

FIG. 1 shows a plot of the relative antibody concentration againstneutralization data for each well. The data was used to identify wellswith the highest potency antibodies.

FIG. 2 is a plot depicting the relationship of ELISA OD of each antibodyversus antibody concentration at an antigen coating of 31 ng/mL.

FIG. 3 is a graph showing the percent inhibition of IL-13 inducedeotaxin release by recombinant antibodies 643 and 731 compared to anisotype matched control.

FIG. 4 is a bar graph comparing the ability of IL-13 or IL-13Q110R toinhibit binding of 731 or 623 to IL-13 coated ELISA plates.

FIG. 5A is a bar graph comparing on cell receptor competition betweenantibody 643 and an isotype control.

FIG. 5B is a bar graph comparing on cell receptor competition betweenantibody 731 and an isotype control.

FIG. 5C is a cartoon depicting the protocol and various predictedresults from FIG. 5A and FIG. 5E.

FIG. 5D is a cartoon depicting the protocol and various predictedresults from FIG. 5B.

FIG. 5E is a bar graph comparing cell receptor competition betweenantibody 623 and an isotype control.

FIG. 6A shows the alignment of a phage-display derived peptiderecognized by antibody 693 and part of IL-13 sequence.

FIG. 6B is a chart showing the secondary structure of IL-13 (SEQ ID NO:72) and indicates which regions of human IL-13 were replaced with mouseIL-13 for the construction of the chimeric proteins.

FIG. 7A and FIG. 7B are bar graphs showing that CD4⁺ T cells fromhumanized IL-13 mice produce human IL-13 but not murine IL-13.

FIG. 8 is a graph demonstrating that anti-IL-13 antibodies 731 and 623inhibit airway hyperresponsiveness.

FIG. 9 is a bar graph demonstrating that 731 and 623 inhibit mucusproduction.

FIG. 10 is a depiction of an amino acid sequence highlighting a bindingsite of mAb 623.

FIGS. 11A-D are graphs displaying the percent inhibition of the eotaxinrelease induced by IL-13 or IL-13Q110R variant by recombinant antibodies623 and 731 compared to an isotype matched control.

FIGS. 12A and 12B are graphs demonstrating inhibition of L-1236 (A) andHDLM-2 (B) cell line proliferation by 623 and 731. mAb 623, mAb 731 orisotype matched control were added to the plate for final concentrationsof 0.017 to 330 nM (titrated 1:3).

FIG. 13 is a graph displaying the impact of mAb 623 and 731 andhIL-13Ralpha2Fc on CD23 expression on whole blood B cells.

FIG. 14 is a graph displaying the inhibition of OVA-Induced mucusproduction by mAb 623 and mAb 731 in IL-13 humanized mice.

FIG. 15 is a graph displaying an experiment in which treatment with mAb623 or 731 had little observable effect on OVA-induced leukocyterecruitment in BALF.

FIG. 16 is graph displaying the inhibition of OVA-induced AHR by 623 and731 in IL-13 humanized mice in a dosage responsive manner.

FIG. 17 is a graph displaying inhibition of OVA-induced mucus productionby 623 and 731 in IL-13 humanized mice in a dosage responsive manner.

FIG. 18 is a graph displaying an effect of 623 and 731 on OVA-inducedleukocyte infiltration in the BALF of IL-13 humanized mice in a dosageresponsive manner.

FIG. 19 is a graph displaying a dose response inhibition of HDM-InducedAHR by 623 and 731 Dose Response in IL-13 humanized mice.

FIG. 20 is a graph displaying a dose response inhibition of HDM-inducedmucus production by 623 and 731 in IL-13 humanized mice.

FIG. 21 is a graph displaying a dose response inhibition of 623 and 731on HDM-induced leukocyte infiltration in the BALF of IL-13 humanizedmice.

FIGS. 22A and 22B are graphs depicting the effect of prophylactic andtherapeutic 623 administration on HDM-induced AHR and leukocyteinfiltration in the BALF of IL-13 humanized mice.

FIG. 23 is a graph depicting OVA-induced serum levels of TARC in wildtype mice.

FIG. 24 is a graph depicting OVA-induced serum levels of eotaxin in wildtype mice.

FIG. 25 is a graph depicting OVA-induced serum levels of C10 in wildtype mice.

FIG. 26 is a graph depicting mAb 623 inhibition of OVA-induced serumlevels of TARC in IL-13 humanized mice.

FIG. 27 is a graph depicting mAb 623 inhibition of OVA-induced serumlevels of eotaxin in IL-13 humanized mice.

FIG. 28 is a graph depicting mAb the effect of mAb 623 treatment onOVA-induced serum levels of C10 in IL-13 humanized mice.

DETAILED DESCRIPTION

Some embodiments of the invention relate to isolated antibodies thatbind to IL-13 and methods of using those antibodies to treat diseases inhumans. Preferably the antibodies are fully human monoclonal antibodiesthat bind to IL-13 with high affinity, high potency, or both. In oneembodiment, the antibodies, or antibody fragments, specifically bind toregions of the IL-13 molecule that prevent it from signaling through theIL-13 receptor complex. In one embodiment, the fully human monoclonalantibodies are neutralizing towards IL-13 based activity.

In another embodiment of the invention, the antibodies bind to IL-13while allowing it to bind to a receptor, other than the IL-13 receptoralpha 1. For example, in one embodiment, the antibody binds to IL-13 andallows IL-13 to bind to a decoy receptor known as IL-13 receptor alpha2. In this case, the antibody prevents IL-13 from binding to itssignaling receptor, but not to the decoy receptor. Embodiments of theinvention also include cells for producing these antibodies.

In addition, embodiments of the invention include methods of using theseanti-IL-13 antibodies as a diagnostic agent or treatment for a disease.For example, the antibodies are useful for treating asthma, includingboth allergic (atopic) and non-allergic (non-atopic), bronchial asthma,chronic bronchitis, emphysema, chronic obstructive pulmonary disease(COPD), hay fever, rhinitis, urticaria, angioedema, allergic dermatitis,including contact dermatitis, Stevens-Johnson syndrome, anaphylatcticshock, food allergies, keratitis, conjunctivitis, steroid-resistantnephritic syndrome, mastocytosis, fibrotic disease such as lungfibrosis, including idiopathic pulmonary fibrosis, cystic fibrosis,bleomycin-induced fibrosis, hepatic fibrosis and systemic sclerosis,cancers, such as Hodgkin's disease, B-cell proliferative disorders suchas B-cell lymphoma, particularly mediastinal large B-cell lymphoma,B-cell leukemias, ovarian carcinoma, diseases characterized bynon-malignant B-cell proliferation such as systemic lupus erythematosus,rheumatoid arthritis, chronic active hepatitis and cryoglobulinemias,high levels of autoantibodies, such as hemolytic anemia,thrombocytopenia, phospholipids syndrome and pemphigus, inflammatorybowel disease and graft-versus-host disease. In some embodiments, themethod of treatment further comprises checking the effectiveness of theadministration of the antibody by following the level of a biomarker,such as eotaxin, TARC and/or C10.

In association with such treatment, embodiments of the invention includearticles of manufacture comprising the antibodies. One embodiment of theinvention is an assay kit comprising IL-13 antibodies that is used toscreen for diseases or disorders associated with IL-13 activity. In someembodiments, the kit includes a biomarker, allowing one to determine theeffectiveness of the antibody in a particular patient.

Additionally, antibodies to IL-13 have been used to influenceinterleukin-13's (IL-13) role as an effector cytokine, which plays arole in the pathogenesis of asthma and other disorders. In animals,direct administration of IL-13 induces asthma, and blockade of IL-13inhibits IL-13-induced or allergen-induced asthma. As shown herein, mAb623 binds with high affinity to IL-13 (K_(D)=24 pM) and IL-13R130Q(K_(D)=39 pM), a common IL-13 variant associated with allergy andasthma. Furthermore, it is presently shown that mAb 623 prevents IL-13from binding to IL-13Rα1 and IL-13Rα2. In vitro, mAb 623 inhibitsIL-13-induced eotaxin-1 production by human dermal fibroblast (HDFa)cells, and IL-13-induced CD23 up-regulation on whole blood B cells.Additionally, mAb 623 also inhibits cell proliferation of HDLM-2 andL-1236 cells, two Hodgkin's lymphoma-derived cell lines that use IL-13as an autocrine growth factor. Thus, the antibody appears to have a widerange of desirable characteristics relating to treating disordersrelating to IL-13.

Additionally, a mouse model designed for examining asthma has also beendeveloped and tested. As mAb 623 does not bind to murine IL-13, IL-13KI/KO mice were generated by replacing the first exon of the murineIL-13 gene with the cDNA encoding human IL-13, thereby allowing humanIL-13 to be expressed under the murine IL-13 promoter and removing theexpression of the endogenous murine IL-13 gene. Using these mice toestablish an OVA-induced asthma model, it is herein shown thatprophylactic administration of mAb 623 blocks airway hyperreactivity(AHR) and significantly suppresses mucus hyperplasia. Furthermore, in ahouse dust mite-induced asthma model, prophylactic or therapeuticadministration of mAb 623 inhibits AHR, mucus hyperplasia and eosinophilinfiltration in the airways.

Additionally, mAb 623 also inhibits OVA-induced elevation of TARC andeotaxin-1 serum levels, demonstrating that these compounds can be usefulas biomarkers. Additionally, these data show that mAb 623 and otherantibodies can effectively neutralize IL-13 in vitro and in vivo. Theantibodies disclosed herein, as well as those created from the disclosedmethods, can also be used and can exhibit similar properties. Methodsfor screening and verifying the particular properties of the antibodiesare provided herein.

The nucleic acids described herein, and fragments and variants thereof,may be used, by way of nonlimiting example, (a) to direct thebiosynthesis of the corresponding encoded proteins, polypeptides,fragments and variants as recombinant or heterologous gene products, (b)as probes for detection and quantification of the nucleic acidsdisclosed herein, (c) as sequence templates for preparing antisensemolecules, and the like. Such uses are described more fully below.

In some aspects, methods of identifying these antibodies are provided.In one embodiment, the method involves an eotaxin release assay.

In some aspects, antibodies that bind to a variant of IL-13 are alsoprovided. Especially relevant are those antibodies that bind to an IL-13variant with a Glutamine at position 110 of the endogenous IL-13polypeptide.

In some aspects, a mouse that is genetically altered to produce onlyhuman IL-13 is provided. This mouse is useful for providing a testsubject for airway hyperresponsiveness and inhibition of mucusproduction.

In some aspects, the antibodies can be used for the prophylactictreatment or prevention of asthma or any of the herein discloseddisorders. For example, in some embodiments, the antibody can be used toprophylactically treat any of the following: inflammatory diseases,cancer, fibrotic disease and diseases characterized by non-malignantcell proliferation; inflammatory diseases or disorders such as asthma,including both allergic (atopic) and non-allergic (non-atopic),bronchial asthma, chronic bronchitis, emphysema, chronic obstructivepulmonary disease (COPD), hay fever, rhinitis, urticaria, angioedema,allergic dermatitis, including contact dermatitis, Stevens-Johnsonsyndrome, anaphylactic shock, food allergies, keratitis, conjunctivitis,steroid-resistant nephritic syndrome; mastocytosis; fibrotic diseasesuch as lung fibrosis, including idiopathic pulmonary fibrosis, cysticfibrosis, bleomycin-induced fibrosis, hepatic fibrosis and systemicsclerosis. In further embodiments the anti-IL-13 antibodies are used totreat cancers, such as Hodgkin's disease, B-cell proliferative disorderssuch as B-cell lymphoma, particularly mediastinal large B-cell lymphoma,B-cell leukemias, ovarian carcinoma. The antibody can be administeredbefore or during any risk of the disease. In some embodiments, theantibody is given in a single dose or multiple doses. In someembodiments, the antibody is administered continuously. In chronicconditions, the antibody can be administered in larger doses and/orcontinuously. In acute conditions, the antibody can be administered in asingle or low dose, or relatively infrequently.

In some embodiments, the methods disclosed herein can be used foridentifying biomarkers for a disease or biological events relating to orimpacting IL-13. In some embodiments, the biomarker is selected from thegroup consisting of: C10, TARC, eotaxin, and some combination thereof.In some embodiments, one monitors the level of C10, TARC and/or eotaxinin a subject that can benefit from the monitoring of a biomarkerrelating to IL-13. In some embodiments, one administers the antibody toa patient, e.g., mAb 623, and then monitors the level of the biomarkerto verify the effectiveness of the antibody. In some embodiments, onethen adjusts the amount of antibody administered to the patient. In someembodiments, molecules that bind to and detect these biomarkers(“biomarker detectors”), and their use to detect the biomarkers, inconnection with determining the effectiveness of treating an IL-13related disorder is contemplated. For example, antibodies to thesemarkers are also useful for the detection of the biomarkers.

Definitions:

Unless otherwise defined, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular.

Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art, as described in various generaland more specific references such as those that are cited and discussedthroughout the present specification. See e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2^(nd) ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al. Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by reference.Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. Standard techniques arealso used for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.

As utilized in accordance with the present disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings:

“Polymerase chain reaction” or “PCR” refers to a procedure or techniquein which minute amounts of a specific piece of nucleic acid, RNA and/orDNA, are amplified as described in U.S. Pat. No. 4,683,195 issued Jul.28, 1987. Generally, sequence information from the ends of the region ofinterest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical or similar insequence to opposite strands of the template to be amplified. The 5′terminal nucleotides of the two primers can coincide with the ends ofthe amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol.51:263 (1987); Erlich, ed., PCR Technology (Stockton Pres, NY, 1989). Aused herein, PCR is considered to be one, but not the only, example of anucleic acid polymerase reaction method for amplifying a nucleic acidtest sample comprising the use of a known nucleic acid as a primer and anucleic acid polymerase to amplify or generate a specific piece ofnucleic acid.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules which lack antigenspecificity. Polypeptides of the latter kind are, for example, producedat low levels by the lymph system and at increased levels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains (Chothia et al. J. Mol.Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A.82:4592 (1985); Chothia et al., Nature 342:877-883 (1989)).

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments including Fab andF(ab)′2, so long as they exhibit the desired biological activity. The“light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, called κand λ, based on the amino acid sequences of their constant domains.Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact antibodies. Binding fragmentsinclude Fab, Fab′, F(ab′)₂, Fv, and single-chain antibodies, asdescribed in more detail below. An antibody other than a “bispecific” or“bifunctional” antibody is understood to have each of its binding sitesidentical.

Preferred antibodies are neutralizing and inhibit binding of IL-13 to asignaling receptor, such as IL-13 receptor alpha-1 (IL-13Rα1) by atleast about 20%, 40%, 60% or 80%, and more usually greater than about85% (as measured in an in vitro competitive binding assay). In someembodiments the antibodies also inhibit binding to the decoy receptorIL-13Rα2, while in other embodiments the ability of IL-13 to bindIL-13Rα2 is maintained upon antibody binding to IL-13.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and terminal or internal amino acid sequence by use ofa spinning cup sequenator, or (2) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

A “neutralizing antibody” is an antibody molecule that is able toeliminate or significantly reduce an effector function of a targetantigen to which it binds. Accordingly, a “neutralizing” IL-13 antibodyis capable of eliminating or significantly reducing an effectorfunction, such as IL-13 signaling activity through the IL-13 receptor.In one embodiment, a neutralizing antibody will reduce an effectorfunction by 1-10, 10-20, 20-30, 30-50, 50-70, 70-80, 80-90, 90-95,95-99, 99-100%.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which non-specific cytotoxic cells thatexpress Ig Fc receptors (FcRs) (e.g. Natural Killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. FcRs expression on hematopoieticcells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu.Rev. Immunol. 9:457-492 (1991). To assess ADCC activity of a molecule ofinterest, an in vitro ADCC assay, such as that described in U.S. Pat.No. 5,500,362, or 5,821,337 may be performed. Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al. PNAS (USA) 95:652-656(1988).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the Ig light-chain and heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al. (1991)). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

Digestion of antibodies with the enzyme, papain, results in twoidentical antigen-binding fragments, known also as “Fab” fragments, anda “Fc” fragment, having no antigen-binding activity but having theability to crystallize. Digestion of antibodies with the enzyme, pepsin,results in the a F(ab′)₂ fragment in which the two arms of the antibodymolecule remain linked and comprise two-antigen binding sites. TheF(ab′)₂ fragment has the ability to crosslink antigen.

“Fv” when used herein refers to the minimum fragment of an antibody thatretains both antigen-recognition and antigen-binding sites.

“Fab” when used herein refers to a fragment of an antibody thatcomprises the constant domain of the light chain and the CH1 domain ofthe heavy chain.

“Fv” is the minimum antibody fragment that contains a completeantigen-binding recognition and binding site. In a two-chain Fv species,this region consists of a dimer of one heavy- and one light-chainvariable domain in tight, non-covalent association. In a single-chain Fvspecies, one heavy- and one light-chain variable domain can becovalently linked by a flexible peptide linker such that the light andheavy chains can associate in a “dimeric” structure analogous to that ina two-chain Fv species. It is in this configuration that the three CDRsof each variable domain interact to define an antigen-binding site onthe surface of the VH-VL dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g. residues 24-34 (L1),50-62 (L2), and 89-97 (L3) in the light chain variable domain and 31-55(H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domainKabat et al., Sequences of Proteins of Immunological Interest, 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991)) and/or those residues from a “hypervariable loop” (e.g. residues26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domainand 26-32 ((H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). “FrameworkRegion” or “FR” residues are those variable domain residues other thanthe hypervariable region residues as herein defined.

The term “complementarity determining regions” or “CDRs” when usedherein refers to parts of immunological receptors that make contact witha specific ligand and determine its specificity. The CDRs ofimmunological receptors are the most variable part of the receptorprotein, giving receptors their diversity, and are carried on six loopsat the distal end of the receptor's variable domains, three loops comingfrom each of the two variable domains of the receptor.

The term “epitope” is used to refer to binding sites for antibodies onprotein antigens. Epitopic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. An antibodyis said to bind an antigen when the dissociation constant is ≦1 μM,preferably ≦100 nM and most preferably ≦10 nM. An increased or greaterequilibrium constant (“K_(D)”) means that there is less affinity betweenthe epitope and the antibody. In other words, that the antibody and theepitope are less favorable to bind or stay bound together. A decreasedor lower equilibrium constant means that there is a higher affinitybetween the epitope and the antibody. In other words, it is more likelythat the antibody and the epitope will bind or stay bound together. Anantibody with a K_(D) of “no more than” a certain amount means that theantibody will bind to the epitope with the given K_(D), or more strongly(or tightly). In some embodiments, the antibody binds with a K_(D) of nomore than 200 pm, for example, 200-180, 180-170, 170-60, 160-150,150-140, 140-130, 130-120, 12-100, 100-80, 80-60, 60-50, 50-40, 40-30,30-20, 20-10, 10-1,1-0.1 pM or less.

While K_(D) describes the binding characteristics of an epitope and anantibody, “potency” describes the effectiveness of the antibody itselffor a function of the antibody. A relatively low K_(D) does notautomatically mean a high potency. Thus, antibodies can have arelatively low K_(D) and a high potency (e.g., they bind well and alterthe function strongly), a relatively high K_(D) and a high potency(e.g., they don't bind well but have a strong impact on function), arelatively low K_(D) and a low potency (e.g., they bind well, but not ina manner effective to alter a particular function) or a relatively highK_(D) and a low potency (e.g., they simply do not bind to the targetwell). In one embodiment, high potency means that there is a high levelof inhibition with a low concentration of antibody. In one embodiment,an antibody is potent or has a high potency when its IC₅₀ is a smallvalue, for example, 130-110, 110-90, 90-60, 60-30, 30-25, 25-20, 20-15,or less pM.

“Substantially,” unless otherwise specified in conjunction with anotherterm, means that the value can vary within the any amount that iscontributable to errors in measurement that may occur during thecreation or practice of the embodiments. “Significant” means that thevalue can vary as long as it is sufficient to allow the claimedinvention to function for its intended use.

The term “selectively bind” in reference to an antibody does not meanthat the antibody only binds to a single substance. Rather, it denotesthat the K_(D) of the antibody to a first substance is less than theK_(D) of the antibody to a second substance. Antibodies that exclusivelybind to an epitope only bind to that single epitope.

The term “amino acid” or “amino acid residue,” as used herein, refers tonaturally occurring L amino acids or to D amino acids as describedfurther below with respect to variants. The commonly used one andthree-letter abbreviations for amino acids are used herein (BruceAlberts et al., Molecular Biology of the Cell, Garland Publishing, Inc.,New York (3d ed. 1994)).

The term “and/or” denotes 1) including all of the relevant options, 2)including only one (or a subset) of a number of alternative options, 3)including both of the previous descriptions (1) or 2)), and 4) includingonly one of the previous descriptions (1) or 2)).

The term “mAb” refers to monoclonal antibody.

The term “XENOMOUSE® refers to strains of mice which have beenengineered to contain 245 kb and 190 kb-sized germline configurationfragments of the human heavy chain locus and kappa light chain locus, asdescribed in Green et al. Nature Genetics 7:13-21 (1994), incorporatedherein by reference. The XENOMOUSE® strains are available from Abgenix,Inc. (Fremont, Calif.).

The term “XENOMAX®” refers use of to the use of the “Selected LymphocyteAntibody Method” (Babcook et al., Proc. Natl. Acad. Sci. USA,93:7843-7848 (1996)), when used with XENOMOUSE® animals.

The term “SLAM®” refers to the “Selected Lymphocyte Antibody Method”(Babcook et al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996), andSchrader, U.S. Pat. No. 5,627,052), both of which are incorporated byreference in their entireties.

The terms “disease,” “disease state” and “disorder” refer to aphysiological state of a cell or of a whole mammal in which aninterruption, cessation, or disorder of cellular or body functions,systems, or organs has occurred.

The term “symptom” means any physical or observable manifestation of adisorder, whether it is generally characteristic of that disorder ornot. The term “symptoms” can mean all such manifestations or any subsetthereof.

The term “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures, wherein the object is to preventor slow down (lessen) an undesired physiological change or disorder,such as the development or spread of cancer or asthma. “Prophylactictreatment” includes occurrences when a treatment decreases thelikelihood a subject will become sick or increases the amount of timerequired for the subject to become sick or exhibit symptoms orconditions associated with the disorder. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented. The term “inhibit,” when usedin conjunction with a disease or symptom can mean that the antibody canreduce or eliminate the disease or symptom. Prophylactic treatment neednot completely prevent the disease or symptoms. In some embodiments, itdelays the onset of the disease. In other embodiments, it reduces theintensity of the disease or symptoms. In some embodiments, the reductioncan be any amount, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60,60-70, 70-80, 80-90, 90-95, 95-99, 99-100% reduction. The term treatmentcan include similar amounts of improvement or recovery as well.

The term “patient” includes human and veterinary subjects.

“Administer,” for purposes of treatment, means to deliver to a patient.For example and without limitation, such delivery can be intravenous,intraperitoneal, by inhalation, intramuscular, subcutaneous, oral,topical, transdermal, or surgical.

“Therapeutically effective amount,” for purposes of treatment, means anamount such that an observable change in the patient's condition and/orsymptoms could result from its administration, either alone or incombination with other treatment. As discussed herein, and as will beappreciated by one of skill in the art, there are a variety of ways inwhich an effective amount can be determined. For example, an effectiveamount can be an amount required to reduce the amount of a biomarker byany significant amount, including, for example, 0-1, 1-5, 5-10, 10-20,20-30, 30-40, 40-50, 505-60, 60-70, 70-80, 80-90, 90-95, 95-99, 99-100%of a reduction in the biomarker. Alternatively, the amount can be anamount required to reduce a similar percent of the amount of IL-13present in a subject.

An “IL-13 related disorder” is any disease, disorder, or similar suchterm in which IL-13 regulates or influences the disease, optionallyincluding the symptoms of the disease. Examples include inflammatorydiseases, cancer, fibrotic disease diseases characterized bynon-malignant cell proliferation, asthma, including both allergic(atopic) and non-allergic (non-atopic), bronchial asthma, chronicbronchitis, emphysema, chronic obstructive pulmonary disease (COPD), hayfever, rhinitis, urticaria, angioedema, allergic dermatitis, includingcontact dermatitis, Stevens-Johnson syndrome, anaphylactic shock, foodallergies, keratitis, conjunctivitis, steroid-resistant nephriticsyndrome, mastocytosis fibrotic disease such as lung fibrosis, includingidiopathic pulmonary fibrosis, cystic fibrosis, bleomycin-inducedfibrosis, hepatic fibrosis and systemic sclerosis, cancers, such asHodgkin's disease, B-cell proliferative disorders such as B-celllymphoma, particularly mediastinal large B-cell lymphoma, B-cellleukemias, ovarian carcinoma, diseases characterized by non-malignantB-cell proliferation such as systemic lupus erythematosus, rheumatoidarthritis, chronic active hepatitis and cryoglobulinemias; diseasecharacterized by high levels of autoantibodies, such as hemolyticanemia, thrombocytopenia, phospholipids syndrome and pemphigus;inflammatory bowel disease; and graft-versus-host disease. In someembodiments, an “IL-13 ligand dependent disorder” is any of the abovethat can be directly influenced by the binding of an antibody to IL-13.In other words, the disorder is directly the result of excessive amountsof IL-13. In some embodiments, an IL-13 antibody treatable disorder isany of the above that can be effectively treated by the addition of oneof the presently disclosed antibodies. Altering “IL-13 related activity”can include treating any of the above disorders with an antibody; it canalso include other, nontherapeutic or prophylactic uses of the antibodywhich may alter the activity of IL-13. In some embodiments, “IL-13related disorder” can encompass any disorder in which an elevated levelof IL-13 is present in the patient. In some embodiments, “IL-13 relateddisorder” can encompass any disorder that has a phenotype that ischaracteristic of IL-13. Phenotypes that are characteristic of a patientwith an IL-13 related disorder can be determined and observed byadministering an amount of IL-13 to a patient to induce variousphenotypes. The amount of IL-13 administered can vary and can beroutinely determined by one of skill in the art. In some embodiments,and IL-13 related disorder is one which is a TH2 cytokine mediated orrelated disorder.

As used herein, the term “biomarker” can encompass any molecule that cantrack or follow the level of IL-13 related activity or concentration ina sample. Examples of IL-13 biomarkers include C10, TARC, and eotaxin. A“biomarker detector” is any molecule or technique, which allows one todetermine the amount, and in some embodiments, the change in the amount,of the biomarker in a sample. For example, antibodies to the biomarker,ligands or receptors to the biomarker, various small peptides that bindto the receptor would all be included as types of biomarker detectors.

A “pharmaceutically acceptable vehicle,” for the purposes of treatment,is a physical embodiment that can be administered to a patient.Pharmaceutically acceptable vehicles can be, but are not limited to,pills, capsules, caplets, tablets, orally administered fluids,injectable fluids, sprays, aerosols, lozenges, neutraceuticals, creams,lotions, oils, solutions, pastes, powders, vapors, or liquids. Oneexample of a pharmaceutically acceptable vehicle is a buffered isotonicsolution, such as phosphate buffered saline (PBS).

“Neutralize,” for purposes of treatment, means to partially orcompletely suppress chemical and/or biological activity.

“Down-regulate,” for purposes of treatment, means to lower the level ofa particular target composition.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as monkeys, dogs, horses, cats, cows, etc.

The term “polynucleotide” as referred to herein means a polymeric formof nucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide. Theterm includes single and double stranded forms of DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide of genomic, cDNA, or synthetic origin or some combinationthereof, which by virtue of its origin the “isolated polynucleotide” (1)is not associated with all or a portion of a polynucleotide in which the“isolated polynucleotide” is found in nature, (2) is operably linked toa polynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence.

The term “oligonucleotide” referred to herein includes naturallyoccurring, and modified nucleotides linked together by naturallyoccurring, and non-naturally occurring oligonucleotide linkages.Oligonucleotides are a polynucleotide subset generally comprising alength of 200 bases or fewer. Preferably, oligonucleotides are 10 to 60bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or20 to 40 bases in length. Oligonucleotides are usually single stranded,e.g., for probes; although oligonucleotides may be double stranded,e.g., for use in the construction of a gene mutant. Oligonucleotides canbe either sense or antisense oligonucleotides.

The term “naturally occurring nucleotide” as used herein includesdeoxyribonucleotides and ribonucleotides. The term “modifiednucleotides” referred to herein includes nucleotides with modified orsubstituted sugar groups and the like. The term “oligonucleotidelinkages” referred to herein includes oligonucleotides linkages such asphosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. AcidsRes. 14:9081 (1986); Stec et al. J. Am. Chem. Soc. 106:6077 (1984);Stein et al. Nucl. Acids Res. 16:3209 (1988); Zon et al. Anti-CancerDrug Design 6:539 (1991); Zon et al. Oligonucleotides and Analogues: APractical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford UniversityPress, Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures ofwhich are hereby incorporated by reference. An oligonucleotide caninclude a label for detection, if desired.

The term “selectively hybridize” referred to herein means to detectablyand specifically bind. Polynucleotides, oligonucleotides and fragmentsthereof selectively hybridize to nucleic acid strands underhybridization and wash conditions that minimize appreciable amounts ofdetectable binding to nonspecific nucleic acids. High stringencyconditions can be used to achieve selective hybridization conditions asknown in the art and discussed herein. Generally, the nucleic acidsequence homology between the polynucleotides, oligonucleotides, orantibody fragments and a nucleic acid sequence of interest will be atleast 80%, and more typically with preferably increasing homologies ofat least 85%, 90%, 95%, 99%, and 100%.

The term “control sequence” as used herein refers to polynucleotidesequences which are necessary to effect the expression and processing ofcoding sequences to which they are connected. The nature of such controlsequences differs depending upon the host organism; in prokaryotes, suchcontrol sequences generally include promoter, ribosomal binding site,and transcription termination sequence; in eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, all components whose presence is essential for expression andprocessing, and can also include additional components whose presence isadvantageous, for example, leader sequences and fusion partnersequences.

The term “operably linked” as used herein refers to positions ofcomponents so described that are in a relationship permitting them tofunction in their intended manner. For example, a control sequence“operably linked” to a coding sequence is connected in such a way thatexpression of the coding sequence is achieved under conditionscompatible with the control sequences.

The term “isolated protein” referred to herein means a protein of cDNA,recombinant RNA, or synthetic origin or some combination thereof, whichby virtue of its origin, or source of derivation, the “isolated protein”(1) is not associated with proteins found in nature, (2) is free ofother proteins from the same source, e.g., free of murine proteins, (3)is expressed by a cell from a different species, or (4) does not occurin nature.

The term “polypeptide” is used herein as a generic term to refer tonative protein, fragments, or analogs of a polypeptide sequence. Hence,native protein, fragments, and analogs are species of the polypeptidegenus. Preferred polypeptides in accordance with the invention comprisethe human heavy chain immunoglobulin molecules represented by SEQ IDNOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, and81-88, for example, and the human kappa light chain immunoglobulinmolecules represented by SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36,40, 44, 48, 52, 56, 60, and 89-94, for example, as well as antibodymolecules formed by combinations comprising the heavy chainimmunoglobulin molecules with light chain immunoglobulin molecules, suchas the kappa light chain immunoglobulin molecules, and vice versa, aswell as fragments and analogs thereof. In some embodiments, the antibodyhas a sequence from the heavy chain CDR1, CDR2, CDR3, FR1, FR2, FR3,and/or FR4 or any of the sequences listed in Table 18. In someembodiments, the antibody has a sequence from the light chain CDR1,CDR2, CDR3, FR1, FR2, FR3, and/or J or any of the sequences listed intables 19 and 20.

Unless specified otherwise, the left-hand end of single-strandedpolynucleotide sequences is the 5′ end; the left-hand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ to the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred to as“downstream sequences”.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)), which is incorporated herein by reference.Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as alpha-, alpha-disubstituted aminoacids, N-alkyl amino acids, lactic acid, and other unconventional aminoacids may also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence.

In contradistinction, the term “complementary to” is used herein to meanthat the complementary sequence is homologous to all or a portion of areference polynucleotide sequence. For illustration, the nucleotidesequence “TATAC” corresponds to a reference sequence “TATAC” and iscomplementary to a reference sequence “GTATA”.

The following terms are among those used to describe the sequencerelationships between two or more polynucleotide or amino acidsequences: “reference sequence”, “comparison window”, “sequenceidentity”, “percentage of sequence identity”, “substantial identity”,and “homology.” A “reference sequence” is a defined sequence used as abasis for a sequence comparison. A reference sequence may be a subset ofa larger sequence, for example, as a segment of a full-length cDNA orgene sequence given in a sequence listing or may comprise a completecDNA or gene sequence. Generally, a reference sequence is at least 18nucleotides or 6 amino acids in length, frequently at least 24nucleotides or 8 amino acids in length, and often at least 48nucleotides or 16 amino acids in length. Since two polynucleotides oramino acid sequences may each (1) comprise a sequence (i.e., a portionof the complete polynucleotide or amino acid sequence) that is similarbetween the two molecules, and (2) may further comprise a sequence thatis divergent between the two polynucleotides or amino acid sequences,sequence comparisons between two (or more) molecules are typicallyperformed by comparing sequences of the two molecules over a “comparisonwindow” to identify and compare local regions of sequence similarity.

A “comparison window”, as used herein, refers to a conceptual segment ofat least about 18 contiguous nucleotide positions or about 6 amino acidswherein the polynucleotide sequence or amino acid sequence is comparedto a reference sequence of at least 18 contiguous nucleotides or 6 aminoacid sequences and wherein the portion of the polynucleotide sequence inthe comparison window may include additions, deletions, substitutions,and the like (i.e., gaps) of 20 percent or less as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Optimal alignment of sequencesfor aligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch J. Mol. Biol.48:443 (1970), by the search for similarity method of Pearson and LipmanProc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, (Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), GENEWORKS™, or MACVECTOR®software packages), or by inspection, and the best alignment (i.e.,resulting in the highest percentage of homology over the comparisonwindow) generated by the various methods is selected.

The term “sequence identity” means that two polynucleotide or amino acidsequences are identical (i.e., on a nucleotide-by-nucleotide orresidue-by-residue basis) over the comparison window. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) or amino acid residue occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the comparison window(i.e., the window size), and multiplying the result by 100 to yield thepercentage of sequence identity. The terms “substantial identity” asused herein denotes a characteristic of a polynucleotide or amino acidsequence, wherein the polynucleotide or amino acid comprises a sequencethat has at least 85 percent sequence identity, preferably at least 90to 95 percent sequence identity, more preferably at least 99 percentsequence identity, as compared to a reference sequence over a comparisonwindow of at least 18 nucleotide (6 amino acid) positions, frequentlyover a window of at least 24-48 nucleotide (8-16 amino acid) positions,wherein the percentage of sequence identity is calculated by comparingthe reference sequence to the sequence which may include deletions oradditions which total 20 percent or less of the reference sequence overthe comparison window. The reference sequence may be a subset of alarger sequence.

Two amino acid sequences or polynucleotide sequences are “homologous” ifthere is a partial or complete identity between their sequences. Forexample, 85% homology means that 85% of the amino acids are identicalwhen the two sequences are aligned for maximum matching. Gaps (in eitherof the two sequences being matched) are allowed in maximizing matching;gap lengths of 5 or less are preferred with 2 or less being morepreferred. Alternatively and preferably, two protein sequences (orpolypeptide sequences derived from them of at least about 30 amino acidsin length) are homologous, as this term is used herein, if they have analignment score of at more than 5 (in standard deviation units) usingthe program ALIGN with the mutation data matrix and a gap penalty of 6or greater. See Dayhoff, M. O., in Atlas of Protein Sequence andStructure, pp. 101-110 (Volume 5, National Biomedical ResearchFoundation (1972)) and Supplement 2 to this volume, pp. 1-10. The twosequences or parts thereof are more preferably homologous if their aminoacids are greater than or equal to 50% identical when optimally alignedusing the ALIGN program.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity, and mostpreferably at least 99 percent sequence identity. Preferably, residuepositions which are not identical differ by conservative amino acidsubstitutions. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, glutamic-aspartic, and asparagine-glutamine.

As discussed herein, minor variations in the amino acid sequences ofantibodies or immunoglobulin molecules are contemplated as beingencompassed by the present invention, providing that the variations inthe amino acid sequence maintain at least 75%, more preferably at least80%, 90%, 95%, and most preferably 99%. In particular, conservativeamino acid replacements are contemplated. Conservative replacements arethose that take place within a family of amino acids that are related intheir side chains. Genetically encoded amino acids are generally dividedinto families: (1) acidic=aspartate, glutamate; (2) basic=lysine,arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan; and (4) unchargedpolar=glycine, asparagine, glutamine, cysteine, serine, threonine,tyrosine. More preferred families are: serine and threonine arealiphatic-hydroxy family; asparagine and glutamine are anamide-containing family; alanine, valine, leucine and isoleucine are analiphatic family; and phenylalanine, tryptophan, and tyrosine are anaromatic family.

For example, it is reasonable to expect that an isolated replacement ofa leucine with an isoleucine or valine, an aspartate with a glutamate, athreonine with a serine, or a similar replacement of an amino acid witha structurally related amino acid will not have a major effect on thebinding or properties of the resulting molecule, especially if thereplacement does not involve an amino acid within a framework site.Whether an amino acid change results in a functional peptide can readilybe determined by assaying the specific activity of the polypeptidederivative. Assays are described in detail herein.

Fragments or analogs of antibodies or immunoglobulin molecules can bereadily prepared by those of ordinary skill in the art. Preferred amino-and carboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Preferably, computerizedcomparison methods are used to identify sequence motifs or predictedprotein conformation domains that occur in other proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. Bowie et al.Science 253:164 (1991). The foregoing examples demonstrate that those ofskill in the art can recognize sequence motifs and structuralconformations that may be used to define structural and functionaldomains in accordance with the invention.

Preferred amino acid substitutions are those which: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physiocochemical orfunctional properties of such analogs. Analogs can include variousmuteins of a sequence other than the naturally-occurring peptidesequence. For example, single or multiple amino acid substitutions(preferably conservative amino acid substitutions) may be made in thenaturally-occurring sequence (preferably in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts. Aconservative amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W. H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Brandenand J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et at. Nature 354:105 (1991), which are each incorporatedherein by reference.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the naturally-occurring sequence deduced, for example, froma full-length cDNA sequence. Fragments typically are at least 5, 6, 8 or10 amino acids long, preferably at least 14 amino acids long, morepreferably at least 20 amino acids long. In other embodimentspolypeptide fragments are at least 25 amino acids long, more preferablyat least 50 amino acids long, and even more preferably at least 70 aminoacids long.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics”. Fauchere, J. Adv. Drug Res. 15:29(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al. J.Med. Chem. 30:1229 (1987), which are incorporated herein by reference.Such compounds are often developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biochemical property or pharmacological activity), such ashuman-antibody, but have one or more peptide linkages optionallyreplaced by a linkage selected from the group consisting of: —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and—CH₂SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may be used togenerate more stable peptides. In addition, constrained peptidescomprising a consensus sequence or a substantially identical consensussequence variation may be generated by methods known in the art (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). In certain situations, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In someembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

The term “pharmaceutical agent or drug” as used herein refers to achemical compound or composition capable of inducing a desiredtherapeutic effect when properly administered to a patient. Otherchemistry terms herein are used according to conventional usage in theart, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(Parker, S., Ed., McGraw-Hill, San Francisco (1985)), incorporatedherein by reference.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 percent of allmacromolecular species present in the composition, more preferably morethan about 85%, 90%, 95%, and 99%. Most preferably, the object speciesis purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of a single macromolecular species.

Antibody Structure

The basic antibody structural unit is known to comprise a tetramer. Eachtetramer is composed of two identical pairs of polypeptide chains, eachpair having one “light” (about 25 kDa) and one “heavy” chain (about50-70 kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition. The carboxy-terminal portion of each chain definesa constant region primarily responsible for effector function. Humanlight chains are classified as kappa and lambda light chains. Heavychains are classified as mu, delta, gamma, alpha, or epsilon, and definethe antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. (See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)), incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site.

Thus, an intact antibody has two binding sites. Except in bifunctionalor bispecific antibodies, the two binding sites are the same.

The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hyper variable regions,also called complementarity determining regions or CDRs. The CDRs fromthe two chains of each pair are aligned by the framework regions,enabling binding to a specific epitope. From N-terminal to C-terminal,both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4. The assignment of amino acids to each domain is inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987);Chothia et al. Nature 342:878-883 (1989).

A bispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites. Bispecific antibodies can be produced by a variety of methodsincluding fusion of hybridomas or linking of Fab′ fragments. (See, e.g.,Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelnyet al. J. Immunol. 148:1547-1553 (1992)). Production of bispecificantibodies can be a relatively labor intensive process compared withproduction of conventional antibodies and yields and degree of purityare generally lower for bispecific antibodies. Bispecific antibodies donot exist in the form of fragments having a single binding site (e.g.,Fab, Fab′, and Fv).

Human Antibodies and Humanization of Antibodies

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of human antibody functioninto a rodent so that the rodent produces fully human antibodies. Unlessspecifically identified herein, “human” and “fully human” antibodies canbe used interchangeably herein. The term “fully human” can be usefulwhen distinguishing antibodies that are only partially human from thosethat are completely, or fully human.

One method for generating fully human antibodies is through the use ofXENOMOUSE® strains of mice which have been engineered to contain 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus. See Green et al. NatureGenetics 7:13-21 (1994). The XENOMOUSE® strains are available fromAbgenix, Inc. (Fremont, Calif.).

The production of the XENOMOUSE® is further discussed and delineated inU.S. patent application Ser. No. 07/466,008, filed Jan. 12, 1990, Ser.No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297, filed Jul. 24,1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filed Ser. No.08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filed Aug. 27,1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No. 08/376,279,filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995, Ser. No.08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun. 5, 1995,Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun.5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857,filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2, 1996,and Ser. No. 08/759,620, filed Dec. 3, 1996 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoMendez et al. Nature Genetics 15:146-156 (1997) and Green and JakobovitsJ. Exp. Med. 188:483-495 (1998). See also European Patent No., EP 0 463151 B1, grant published Jun. 12, 1996, International Patent ApplicationNo., WO 94/02602, published Feb. 3, 1994, International PatentApplication No., WO 96/34096, published Oct. 31, 1996, WO 98/24893,published Jun. 11, 1998, WO 00/76310, published Dec. 21, 2000. Thedisclosures of each of the above-cited patents, applications, andreferences are hereby incorporated by reference in their entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, anda second constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference in their entireties.

Human anti-mouse antibody (HAMA) responses have also led the industry toprepare chimeric or otherwise humanized antibodies. While chimericantibodies have a human constant region and a murine variable region, itis expected that certain human anti-chimeric antibody (HACA) responseswill be observed, particularly in chronic or multi-dose utilizations ofthe antibody. Thus, it would be desirable to provide fully humanantibodies against multimeric enzymes in order to vitiate concernsand/or effects of HAMA or HACA response.

Preparation of Antibodies

Antibodies, as described herein, were prepared using the XENOMOUSE®technology, as described below. Such mice are capable of producing humanimmunoglobulin molecules and antibodies and are deficient in theproduction of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving the same are disclosed in thepatents, applications, and references referred to herein. In particular,however, a preferred embodiment of transgenic production of mice andantibodies therefrom is disclosed in U.S. patent application Ser. No.08/759,620, filed Dec. 3, 1996 and International Patent Application Nos.WO 98/24893, published Jun. 11, 1998 and WO 00/76310, published Dec. 21,2000, the disclosures of which are hereby incorporated by reference. Seealso Mendez et al. Nature Genetics 15:146-156 (1997), the disclosure ofwhich is hereby incorporated by reference.

Through use of such technology, fully human monoclonal antibodies toIL-13 were produced, as described in detail below. Essentially,XENOMOUSE® lines of mice were immunized with human IL-13, lymphaticcells (such as B-cells) were recovered from mice that expressedantibodies, and the recovered cell lines were fused with a myeloid-typecell line to prepare immortal hybridoma cell lines. These hybridoma celllines were screened and selected to identify hybridoma cell lines thatproduced antibodies specific to the IL-13. Further, provided herein arecharacterization of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

Alternatively, instead of being fused to myeloma cells to generatehybridomas, the recovered cells, isolated from immunized XENOMOUSE®lines of mice, can be screened further for reactivity against theinitial antigen, preferably human IL-13. Such screening includes anELISA with the desired IL-13 protein and functional assays such asIL-13-induced eotaxin-1 production. Single B cells secreting antibodiesthat specifically bind to IL-13 can then be isolated using a desiredIL-13-specific hemolytic plaque assay (Babcook et al., Proc. Natl. Acad.Sci. USA, i93:7843-7848 (1996)). Cells targeted for lysis are preferablysheep red blood cells (SRBCs) coated with IL-13. In the presence of a Bcell culture secreting the immunoglobulin of interest and complement,the formation of a plaque indicates specific IL-13-mediated lysis of thetarget cells.

The single antigen-specific plasma cell in the center of the plaque canbe isolated and the genetic information that encodes the specificity ofthe antibody isolated from the single plasma cell. Usingreverse-transcriptase PCR, the DNA encoding the variable region of theantibody secreted can be cloned. Such cloned DNA can then be furtherinserted into a suitable expression vector, preferably a vector cassettesuch as a pcDNA (Invitrogen, Carlsbad, Calif.), more preferably such apcDNA vector containing the constant domains of immunoglobulin heavy andlight chain. The generated vector can then be transfected into hostcells, preferably CHO cells, and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformants,or amplifying the genes encoding the desired sequences.

Herein, is described the isolation of multiple single plasma cells thatproduce antibodies specific to IL-13. Further, the genetic material thatencoded an antibody that specifically bound IL-13 was isolated, and thatmaterial was introduced into a suitable expression vector and thereaftertransfected into host cells.

In general, antibodies produced by the above-mentioned cell linespossessed fully human IgG1 or IgG2 heavy chains with human kappa lightchains. The antibodies possessed high affinities, typically possessingKD's of from about 10⁻⁹ through about 10⁻¹³ M, when measured by eithersolid phase and solution phase.

As mentioned above, anti-IL-13 antibodies can be expressed in cell linesother than hybridoma cell lines. Sequences encoding particularantibodies can be used for transformation of a suitable mammalian hostcell, such as a CHO cell. Transformation can be by any known method forintroducing polynucleotides into a host cell, including, for examplepackaging the polynucleotide in a virus (or into a viral vector) andtransducing a host cell with the virus (or vector) or by transfectionprocedures known in the art, as exemplified by U.S. Pat. Nos. 4,399,216,4,912,040, 4,740,461, and 4,959,455 (which patents are herebyincorporated herein by reference). The transformation procedure useddepends upon the host to be transformed. Methods for introducingheterologous polynucleotides into mammalian cells are well known in theart and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), and a number of other cell lines. Cell lines ofparticular preference are selected through determining which cell lineshave high expression levels and produce antibodies with IL-13 bindingproperties.

Antibody Sequences

The heavy chain and light chain variable region nucleotide and aminoacid sequences of representative human anti-IL-13 antibodies areprovided in the sequence listing, the contents of which are summarizedin Table 1 below.

TABLE 1 mAb SEQ ID ID No.: Sequence NO: 11.18 Amino acid sequenceencoding the variable region of the heavy chain 2 Nucleotide sequenceencoding the variable region of the heavy chain 1 Amino acid sequenceencoding the variable region of the light chain 4 Nucleotide sequenceencoding the variable region of the light chain 3 353 Amino acidsequence encoding the variable region of the heavy chain 6 Nucleotidesequence encoding the variable region of the heavy chain 5 Amino acidsequence encoding the variable region of the light chain 8 Nucleotidesequence encoding the variable region of the light chain 7 356 Aminoacid sequence encoding the variable region of the heavy chain 10Nucleotide sequence encoding the variable region of the heavy chain 9Amino acid sequence encoding the variable region of the light chain 12Nucleotide sequence encoding the variable region of the light chain 11264 Amino acid sequence encoding the variable region of the heavy chain14 Nucleotide sequence encoding the variable region of the heavy chain13 Amino acid sequence encoding the variable region of the light chain16 Nucleotide sequence encoding the variable region of the light chain15 243 Amino acid sequence encoding the variable region of the heavychain 18 Nucleotide sequence encoding the variable region of the heavychain 17 Amino acid sequence encoding the variable region of the lightchain 20 Nucleotide sequence encoding the variable region of the lightchain 19 157 Amino acid sequence encoding the variable region of theheavy chain 22 Nucleotide sequence encoding the variable region of theheavy chain 21 Amino acid sequence encoding the variable region of thelight chain 24 Nucleotide sequence encoding the variable region of thelight chain 23 176 Amino acid sequence encoding the variable region ofthe heavy chain 26 Nucleotide sequence encoding the variable region ofthe heavy chain 25 Amino acid sequence encoding the variable region ofthe light chain 28 Nucleotide sequence encoding the variable region ofthe light chain 27 183 Amino acid sequence encoding the variable regionof the heavy chain 30 Nucleotide sequence encoding the variable regionof the heavy chain 29 Amino acid sequence encoding the variable regionof the light chain 32 Nucleotide sequence encoding the variable regionof the light chain 31 713 Amino acid sequence encoding the variableregion of the heavy chain 34 Nucleotide sequence encoding the variableregion of the heavy chain 33 Amino acid sequence encoding the variableregion of the light chain 36 Nucleotide sequence encoding the variableregion of the light chain 35 731 Amino acid sequence encoding thevariable region of the heavy chain 38 Nucleotide sequence encoding thevariable region of the heavy chain 37 Amino acid sequence encoding thevariable region of the light chain 40 Nucleotide sequence encoding thevariable region of the light chain 39 693 Amino acid sequence encodingthe variable region of the heavy chain 42 Nucleotide sequence encodingthe variable region of the heavy chain 41 Amino acid sequence encodingthe variable region of the light chain 44 Nucleotide sequence encodingthe variable region of the light chain 43 643 Amino acid sequenceencoding the variable region of the heavy chain 46 Nucleotide sequenceencoding the variable region of the heavy chain 45 Amino acid sequenceencoding the variable region of the light chain 48 Nucleotide sequenceencoding the variable region of the light chain 47 623 Amino acidsequence encoding the variable region of the heavy chain 50 Nucleotidesequence encoding the variable region of the heavy chain 49 Amino acidsequence encoding the variable region of the light chain 52 Nucleotidesequence encoding the variable region of the light chain 51 602 Aminoacid sequence encoding the variable region of the heavy chain 54Nucleotide sequence encoding the variable region of the heavy chain 53Amino acid sequence encoding the variable region of the light chain 56Nucleotide sequence encoding the variable region of the light chain 55785 Amino acid sequence encoding the variable region of the heavy chain58 Nucleotide sequence encoding the variable region of the heavy chain57 Amino acid sequence encoding the variable region of the light chain60 Nucleotide sequence encoding the variable region of the light chain59Antibody Therapeutics

Anti-IL-13 antibodies have therapeutic value for treating symptoms andconditions related to IL-13 activity (e.g., an IL-13 related disorder).IL-13 has been implicated in a wide variety of diseases and disorders,including inflammatory diseases, cancer, fibrotic disease and diseasescharacterized by non-malignant cell proliferation. In specificembodiments, the anti-IL-13 antibodies disclosed herein are used in thetreatment of inflammatory diseases or disorders such as asthma,including both allergic (atopic) and non-allergic (non-atopic),bronchial asthma, chronic bronchitis, emphysema, chronic obstructivepulmonary disease (COPD), hay fever, rhinitis, urticaria, angioedema,allergic dermatitis, including contact dermatitis, Stevens-Johnsonsyndrome, anaphylactic shock, food allergies, keratitis, conjunctivitis,steroid-resistant nephritic syndrome. In other embodiments they are usedto treat mastocytosis. In still other embodiments they are used to treatfibrotic disease such as lung fibrosis, including idiopathic pulmonaryfibrosis, cystic fibrosis, bleomycin-induced fibrosis, hepatic fibrosisand systemic sclerosis. In further embodiments the anti-IL-13 antibodiesare used to treat cancers, such as Hodgkin's disease, B-cellproliferative disorders such as B-cell lymphoma, particularlymediastinal large B-cell lymphoma, B-cell leukemias, ovarian carcinoma.

In still further embodiments the anti-IL-13 antibodies are used to treatdiseases characterized by non-malignant B-cell proliferation such assystemic lupus erythematosus, rheumatoid arthritis, chronic activehepatitis and cryoglobulinemias; disease characterized by high levels ofautoantibodies, such as hemolytic anemia, thrombocytopenia,phospholipids syndrome and pemphigus; inflammatory bowel disease; andgraft-versus-host disease. In some embodiments, the antibodies are usedfor the treatment or prevention of asthma in humans, mice, or otheranimals.

In some embodiments, the use of the antibodies in a medicament for thetreatment of an IL-13 related disorder (a disease, condition, etc.,relating to IL-13) is contemplated. The medicament can contain atherapeutically effective amount of the antibody. In some embodiments,the amount of IL-13 in the medicament is sufficient so that at least onebeneficial result is observed, e.g., a lessening of a symptom. In someembodiments, the amount that is administered removes all of the symptomsof the IL-13 related disorder. In some embodiments, the amount issufficient so that the level of a biomarker decreases in a subject afterthe medicament has been administered. In some embodiments, the amount ofthe antibody administered is about 0.001 to 1000, 0.1 to 160, 0.5 to 50,1 to 10, 1, 3, or 10 mg of antibody/kg of subject. As will beappreciated by one of skill in the art, the actual amount of theantibody can depend upon the particular disorder (e.g., asthma, is itacute or chronic), the method of administration, the frequency ofadministration, the desired result, the characteristics of the patient,and the characteristics of the antibody. The actual amount administeredcan be determined by one of skill in the art, through routineexperimentation, in light of the present disclosure. In someembodiments, a single dose will be sufficient. In other embodiments,multiple or continuous doses can be beneficial. The actual amount andmethod of administration can be determined through the use of, amongother techniques, the biomarkers and examples described herein. Forexample, eotaxin, C10, and/or TARC levels can be monitored to providethe optimal level of effectiveness in treatment of the IL-13 relateddisorder. As will be appreciated by one of skill in the art, the use ofthe antibody in the preparation or manufacture of a medicament caninvolve any of the disclosed antibodies in any amount, sufficient totreat the particular condition it is directed to. Any of the hereindisclosed conditions, or any IL-13 related disorders, can be thecondition to be treated. In some embodiments, the use of the antibody inthe preparation of a medicament is with one of the particularantibodies, such as mAb 731, 643, or 623, or any of the antibodieslisted in Table 1. In some embodiments, the antibody used has a K_(D) ofless than 50 or 10 pM. In some embodiments, the antibody used results ina decrease in mucus production of at least 30%. As will be appreciatedby one of skill in the art, the presently disclosed methods of use canbe employed to create a medicament for the use. In some embodiments, themedicament further comprises an antibody or biomarker detector, to abiomarker. In other aspects, the biomarker detector is used in theproduction of a medicament without the antibody to IL-13. The biomarkerdetector in the medicament can be an antibody or other protein thatspecifically binds to the biomarker.

As will be appreciated by one of skill in the art, the nature of thedisorder can play a role in the amount, frequency, and method ofadministration. For example, in chronic disorders, relatively largeramounts, more potent antibodies, and/or more frequently administereddoses of the antibody may be required. Similarly, in acute disorders,the amount of antibody required for treatment, including prophylaxis,can be relatively less. In subjects in which sensitization is initiallyrequired prior to the challenge, lower amounts of the antibody can bebeneficial compared to the amount required for subjects that arenaturally allergic. In such chronic systems, increased amounts of theantibody, as well as increased frequency of administration can beadvantageous. The exact amount can readily be determined by one of skillin the art, in light of the present disclosure. One of skill in the artwill further appreciate other factors and how to adjust theadministration of the antibody accordingly.

If desired, the isotype of an anti-IL-13 antibody can be switched, forexample to take advantage of a biological property of a differentisotype. For example, in some circumstances it may be desirable for thetherapeutic antibodies against IL-13 to be capable of fixing complementand participating in complement-dependent cytotoxicity (CDC). There area number of isotypes of antibodies that are capable of the same,including, without limitation, the following: murine IgM, murine IgG2a,murine IgG2b, murine IgG3, human IgM, human IgG1, and human IgG3. Itwill be appreciated that antibodies that are generated need notinitially possess such an isotype but, rather, the antibody as generatedcan possess any isotype and the antibody can be isotype switchedthereafter using conventional techniques that are well known in the art.Such techniques include the use of direct recombinant techniques (seee.g., U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see e.g.,U.S. Pat. Nos. 5,916,771 and 6,207,418), among others.

By way of example, the anti-IL-13 antibodies discussed herein are humanantibodies. If an antibody possessed desired binding to IL-13, it couldbe readily isotype switched to generate a human IgM, human IgG1, orhuman IgG3 isotype, while still possessing the same variable region(which defines the antibody's specificity and some of its affinity).Such molecule would then be capable of fixing complement andparticipating in CDC.

In the cell-cell fusion technique, a myeloma or other cell line isprepared that possesses a heavy chain with any desired isotype andanother myeloma or other cell line is prepared that possesses the lightchain. Such cells can, thereafter, be fused and a cell line expressingan intact antibody can be isolated.

Accordingly, as antibody candidates are generated that meet desired“structural” attributes as discussed above, they can generally beprovided with at least certain of the desired “functional” attributesthrough isotype switching.

Biologically active antibodies that bind IL-13 are preferably used in asterile pharmaceutical preparation or formulation to reduce the activityof IL-13. Anti-IL-13 antibodies preferably possess adequate affinity topotently suppress IL-13 activity to within the target therapeutic range.The suppression preferably results from the ability of the antibody tointerfere with the binding of IL-13 to a signaling receptor, such asIL-13Ra1 (also known as, IL-13 Rα1, Rα1, IL-13R alpha 1, IL-13 receptoralpha 1, or other similar terms). In other embodiments the antibody maysuppress IL-13 activity by interfering with the ability of IL-13 tosignal through the receptor, even if it is able to bind. For example,the antibody may prevent interaction of the IL-13Ra1 with a co-receptorthat is necessary for signaling, such as the IL-4 receptor alpha chain.In some embodiments the antibody is able to prevent IL-13 activitythrough a signaling receptor while allowing for IL-13 binding to a decoyreceptor, such as IL-13Ra2. In this case, binding to the decoy receptormay allow clearance of the bound IL-13 and enhance the ability of theantibody to suppress IL-13 activity.

When used for in vivo administration, the antibody formulation ispreferably sterile. This is readily accomplished by any method know inthe art, for example by filtration through sterile filtration membranes.The antibody ordinarily will be stored in lyophilized form or insolution. Sterile filtration may be performed prior to or followinglyophilization and reconstitution.

Therapeutic antibody compositions generally are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having an adapter that allows retrieval of the formulation, suchas a stopper pierceable by a hypodermic injection needle.

The modality of antibody administration is in accord with known methods,e.g., injection or infusion by subcutaneous, intravenous,intraperitoneal, intracerebral, intradermic, intramuscular, intraocular,intraarterial, intrathecal, or intralesional routes, or by inhalation orby sustained release systems as noted below. In some situations theantibody is preferably administered by infusion or by bolus injection.In other situations a therapeutic composition comprising the antibodycan be administered through the nose or lung, preferably as a liquid orpowder aerosol (lyophilized). The composition may also be administeredintravenously, parenterally or subcutaneously as desired. Whenadministered systemically, the therapeutic composition should besterile, pyrogen-free and in a parenterally acceptable solution havingdue regard for pH, isotonicity, and stability. These conditions areknown to those skilled in the art.

Antibodies for therapeutic use, as described herein, are typicallyprepared with suitable carriers, excipients, and other agents that areincorporated into formulations to provide improved transfer, delivery,tolerance, and the like. Briefly, dosage formulations of the antibodiesdescribed herein are prepared for storage or administration by mixingthe antibody having the desired degree of purity with one or morephysiologically acceptable carriers, excipients, or stabilizers. Theseformulations may include, for example, powders, pastes, ointments,jellies, waxes, oils, lipids, lipid (cationic or anionic) containingvesicles (such as Lipofectin™), DNA conjugates, anhydrous absorptionpastes, oil-in-water and water-in-oil emulsions, carbowax (polyethyleneglycols of various molecular weights), semi-solid gels, and semi-solidmixtures containing carbowax. The formulation may include buffers suchas TRIS HCl, phosphate, citrate, acetate and other organic acid salts;antioxidants such as ascorbic acid; low molecular weight (less thanabout ten residues) peptides such as polyarginine, proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidinone; amino acids such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium and/or nonionicsurfactants such as TWEEN, PLURONICS or polyethyleneglycol.

Other acceptable carriers, excipients and stabilizers are well known tothose of skill in the art. Any of the foregoing mixtures may beappropriate in treatments and therapies in accordance with the presentinvention, provided that the active ingredient in the formulation is notinactivated by the formulation and the formulation is physiologicallycompatible and tolerable with the route of administration. See alsoBaldrick P. “Pharmaceutical excipient development: the need forpreclinical guidance.” Regul. Toxicol. Pharmacol. 32(2):210-8 (2000),Wang W. “Lyophilization and development of solid proteinpharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N“Lipids, lipophilic drugs, and oral drug delivery-some emergingconcepts.” J. Pharm. Sci. 89(8):967-78 (2000), Powell et al. “Compendiumof excipients for parenteral formulations” PDA J. Pharm. Sci. Technol.52:238-311 (1998) and the citations therein for additional information.

Sterile compositions for injection can be formulated according toconventional pharmaceutical practice as described in Remington: TheScience and Practice of Pharmacy (20^(th) ed, Lippincott Williams &Wilkens Publishers (2003)). For example, dissolution or suspension ofthe active compound in a vehicle such as water or naturally occurringvegetable oil like sesame, peanut, or cottonseed oil or a syntheticfatty vehicle like ethyl oleate or the like may be desired. Buffers,preservatives, antioxidants and the like can be incorporated accordingto accepted pharmaceutical practice.

The antibodies can also be administered in and released over time fromsustained-release preparations. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the polypeptide. The matrices may be in the form ofshaped articles, films or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (e.g.,poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.Biomed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech., (1982)12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22:547-556),non-degradable ethylene-vinyl acetate (Langer et al., supra), degradablelactic acid-glycolic acid copolymers such as the LUPRON Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid (EP133,988).

While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated proteinsremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for protein stabilization depending on themechanism involved. For example, if the aggregation mechanism isdiscovered to be intermolecular S—S bond formation through disulfideinterchange, stabilization may be achieved by modifying sulfhydrylresidues, lyophilizing from acidic solutions, controlling moisturecontent, using appropriate additives, and developing specific polymermatrix compositions.

Sustained-released compositions also include preparations of crystals ofthe antibody suspended in suitable formulations capable of maintainingcrystals in suspension. These preparations when injected subcutaneouslyor intraperitonealy can produce a sustained release effect. Othercompositions also include liposomally entrapped antibodies. Liposomescontaining such antibodies are prepared by methods known per se: U.S.Pat. No. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. USA,(1985) 82:3688-3692; Hwang et al., Proc. Natl. Acad. Sci. USA, (1980)77:4030-4034; EP 52,322; EP 36,676; EP 88,046; EP 143,949; 142,641;Japanese patent application 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324.

The dosage of the antibody formulation for a given patient may bedetermined by the attending physician. In determining the appropriatedosage the physician may take into consideration various factors knownto modify the action of therapeutics, including, for example, severityand type of disease, body weight, sex, diet, time and route ofadministration, other medications and other relevant clinical factors.Therapeutically effective dosages may be determined by either in vitroor in vivo methods.

An effective amount of the antibodies, described herein, to be employedtherapeutically will depend, for example, upon the therapeuticobjectives, the route of administration, and the condition of thepatient. Accordingly, it is preferred for the therapist to titer thedosage and modify the route of administration as required to obtain theoptimal therapeutic effect. A typical daily dosage might range fromabout 0.001 mg/kg to up to 100 mg/kg or more, depending on the factorsmentioned above. Typically, the clinician will administer thetherapeutic antibody until a dosage is reached that achieves the desiredeffect. The progress of this therapy is easily monitored by conventionalassays.

It is expected that the antibodies described herein will havetherapeutic effect in treatment of symptoms and conditions resultingfrom or related to the activity of IL-13.

One way that the actual dose administered (or used in a kit) can bedetermined or verified is through the identification and use of IL-13dependent biomarkers. Such biomakers are outlined in the Examples belowand FIGS. 23-28, as are methods of identifying the biomarkers and theiruse. Examples of relevant biomarkers include C10, eotaxin, and TARC.Generally, to identify a biomarker, one can measure the quantity of thecandidate biomarker in a subject that has a healthy and/or IL-13disordered state (e.g., OVA-induced asthma), and compare the level ofthe biomarker in the subject to the level of the biomarker when one ofthe effective antibodies is administered to the subject (e.g., mAb 623,731, etc.). As the administration of the antibody can block the activityof IL-13, the level of the candidate biomarker should also decline(assuming it is an actual biomarker) due to the administration of theantibody to the subject.

The biomarker, and/or a method of testing for it, can be used in avariety of ways. For example, the level of the biomarker in a subjectcan be followed throughout the treatment of a subject with an antibody,or other therapeutic method or composition with a similar effect, toallow one to track the effectiveness of the treatment. One can determinethe amount of the biomarker present in the subject initially anddetermine any change upon the addition of the antibody. If the level ofthe biomarker does not change upon the initial administration of theantibody, additional antibody can be applied to the subject, appliedmore frequently, or by an alternative route. One of skill in the artwill appreciate that the level of the biomarker can be determined in avariety of ways and should not unduly limit the technique. Any techniquethat can determine the amount of a protein (or mRNA, etc.) can be used,e.g., ELISA, or Biacore™ techniques. Additionally, in some embodiments,multiple biomarkers can be followed simultaneously. This can allow formore certainty about the IL-13 related aspect being monitored.

Of course, in some embodiments, the biomarkers are simply used to followthe progression of a disorder, without the additional variable ofmonitoring the treatment.

Additionally, the level of the biomarker in a subject can be used todetermine if the subject has an IL-13 related disorder. Subjects withbiomarker levels that are significantly above or below a standard levelor range for a healthy individual(s) could be considered to suffer froman IL-13 related disorder.

The biomarker, or a method of testing for it, can be included in a kitfor the treatment of an IL-13 related disorder.

As will be appreciated by one of skill in the art, while the presentdisclosure extensively discusses conditions or disorders involvingexcessive amounts of IL-13, the compositions and methods could also beapplied to situations in which the effective level of IL-13 is too low.For example, the antibodies do not have to prevent the binding of IL-13to its normal receptor and can instead prevent IL-13 binding to itsdecoy receptor, thereby effectively increasing the amount of IL-13available in the system. However, in many embodiments, the antibodieswill at least prevent IL-13 from binding to it signaling receptor.

Design and Generation of Other Therapeutics

In accordance with the present invention and based on the activity ofthe antibodies that are produced and characterized herein with respectto IL-13, advanced antibody therapeutics may be employed to treatspecific diseases. These advanced therapeutics may include bispecificantibodies, immunotoxins, radiolabeled therapeutics, peptidetherapeutics, gene therapies, particularly intrabodies, antisensetherapeutics, and small molecules.

In connection with the generation of advanced antibody therapeutics,where complement fixation is a desirable attribute, it may be possibleto sidestep the dependence on complement for cell killing through theuse of bispecifics, immunotoxins, or radiolabels, for example.

For example, bispecific antibodies can be generated that comprise (i)two antibodies, one with a specificity to IL-13 and another to a secondmolecule, that are conjugated together, (ii) a single antibody that hasone chain specific to IL-13 and a second chain specific to a secondmolecule, or (iii) a single chain antibody that has specificity to bothIL-13 and the other molecule. Such bispecific antibodies can begenerated using techniques that are well known; for example, inconnection with (i) and (ii) see e.g., Fanger et al. Immunol Methods4:72-81 (1994) and Wright and Harris, supra. and in connection with(iii) see e.g., Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52(1992). In each case, the second specificity can be made as desired. Forexample, the second specificity can be made to the heavy chainactivation receptors, including, without limitation, CD16 or CD64 (seee.g., Deo et al. 18:127 (1997)) or CD89 (see e.g., Valerius et al. Blood90:4485-4492 (1997)).

In some embodiments, an article of manufacture is provided comprising acontainer, comprising a composition containing an anti-IL-13 antibody,and a package insert or label indicating that the composition can beused to treat disease mediated by IL-13. Preferably a mammal and, morepreferably, a human, receives the anti-IL-13 antibody. In preferredembodiments, the disease to be treated is selected from the groupconsisting of asthma, including both allergic (atopic) and non-allergic(non-atopic), bronchial asthma, chronic bronchitis, emphysema, chronicobstructive pulmonary disease (COPD), hay fever, rhinitis, urticaria,angioedema, allergic dermatitis, including contact dermatitis,Stevens-Johnson syndrome, anaphylatctic shock, food allergies,keratitis, conjunctivitis, steroid-resistant nephritic syndrome,mastocytosis, fibrotic disease such as lung fibrosis, includingidiopathic pulmonary fibrosis, cystic fibrosis, bleomycin-inducedfibrosis, hepatic fibrosis and systemic sclerosis, cancers, such asHodgkin's disease, B-cell proliferative disorders such as B-celllymphoma, particularly mediastinal large B-cell lymphoma, B-cellleukemias, ovarian carcinoma, diseases characterized by non-malignantB-cell proliferation such as systemic lupus erythematosus, rheumatoidarthritis, chronic active hepatitis and crioglobulnimias, high levels ofautoantibodies, such as hemolytic anemia, thrombocytopenia,phospholipids syndrome and pemphigus, inflammatory bowel disease andgraft-versus-host disease.

In some embodiments an anti-IL-13 antibody is used to treat asthma. In aparticular embodiment the antibody is the 623 antibody or variantsthereof described herein. In another particular embodiment the antibodyis the 731 antibody or variants thereof described herein. Specificexamples of how these antibodies can be used to treat asthma and otherdisorders are described below in the examples.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the teachings herein.

Example 1 Antibody Generation

IL-13 and IL-13 Antigen Preparation

The following IL-13 peptides were used in the experiments describedbelow.

Recombinant Human IL-13 (R & D 213-IL-005; SEQ ID NO: 61):GPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLL HLKKLFREGQFNRecombinant Human IL-13 (Peprotech 200-13; SEQ ID NO: 62):SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDL LLHLKKLFREGRFNRecombinant Human IL-13 (Peprotech 200-13A; SEQ ID NO: 63):MSPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKD LLLHLKKLFREGQFN HumanIL-13-human Fc fusion protein (with leader sequence; SEQ ID NO: 64):MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK HumanIL-13-rabbit Fc fusion protein (with leader sequence; SEQ ID NO: 65):MALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGRFNRYLDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYNKLSVPTSEWQRGDVFTCSVMHE ALHNHYTQKSISRSPGKHuman IL-13-Mouse IL-13 Helix A (underlined; SEQ ID NO: 66):MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPRSVSLPLTLKELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN Human IL-13-MouseIL-13 Helix B (underlined; SEQ ID NO: 67):MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGGFCVALDSLTNVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN Human IL-13-Mouse IL-13Helix C (underlined; SEQ ID NO: 68):MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIYRTQRILHGLCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN Human IL-13-Mouse IL-13Helix D (underlined; SEQ ID NO: 69):MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAHFITKLLSYTKQLFRHGQQFN

As will be appreciated by one of skill in the art, only a subset of theabove residues may actually be involved in the formation of an epitope.For example, in SEQ ID NOs: 66-69 above, the epitopes may actually bethe helix portion of each peptide (the underlined section). In oneembodiment, the antibodies described herein are directed to any of theIL-13 epitopes or fragments thereof, including the helix portion of eachpeptide.

Immunization of Animals

Monoclonal antibodies against IL-13 were developed by immunizingXenoMouse® mice (XenoMouse® XMG2L3 and XenoMouse® XMG2, Abgenix, Inc.Fremont, Calif.). The human IL-13-human Fc fusion protein (SEQ ID NO:64) or human IL-13-rabbit Fc fusion protein (SEQ ID NO: 65) was used asthe immunogen for antibody generation. Each mouse was immunized via thefootpad route of administration. The animals were immunized on days 0,4, 7, 11, 14, 18, 21 and 25. The initial immunization was with 10 ug ofantigen in CpG/Alum per mouse. Subsequent boosts were with 5 ug ofantigen in CpG/Alum per mouse. The final boost on day 25 was with 5 ugof antigen in PBS without adjuvant per mouse. The animals were bled onday 20 to obtain sera for determination of titer as described below.

Titer Analysis

Titer was determined using a standard protocol. Briefly, Costar 3368plates were coated with either IL-13 rabbit Fc fusion protein (SEQ IDNO: 65) or full length rabbit antibody overnight at 4° C. The plateswere washed using Titertek Program ADG9, dried, and blocked with 250 μl1% no fat skim milk/1×PBS. Following blocking, the plates were washedagain using Titertek Program ADGP and dried. The sera to be tested wastitrated vertically 1:2 in duplicate from a 1:100 initial dilution. Thesamples were run in 1% non fat skim milk/1×PBS at 50 ul/well andincubated for 1 h at room temperature.

After washing using Titertek Program ADG9 and drying, the plates wereincubated for 1 hour at room temperature with a secondary rabbitanti-human Fc antibody conjugated to POD (1:8000 dilution; 50 μL/well)with minimal cross-reactivity to rabbit Fc in 1% no fat skim milk/1×PBS.Plates were then washed a final time using Titertek Program ADG9 anddried. POD substrate one-step TMB solution (50 μl/well) was added andallowed to develop for 30 minutes at room temperature. The reaction wasstopped with 1 N HCL (50 μ/well) and the optical density was readimmediately with a Titertek Plate reader.

Three animals with high titers for the IL-13 immunogen, as shown inTable 2, were selected for harvest.

TABLE 2 Coating Mouse IL-13 Rb Fc RbIgG 1 3855 <100 2 5444 <100 3 >6400268 naïve <100 <100Primary Screen

The hyperimmune animals were harvested and CD19+ B-cells were isolatedfor subsequent B cell culture. The cells were induced to proliferate andterminally differentiate into plasma cells. Supernatants from theseplasma cells were screened by ELISA to identify primary wells containinganti-IL-13-specific antibodies. The cultures were commonly run with 50to 500 CD19+ B cells per well to allow the identification of monoclonalantigen-specific B cell cultures.

Briefly, IL-13-RbFc was coated onto Costar 3368 96 well plates at 1ug/mL overnight. Each plate was washed 5 times with dH₂O and 40 μL of 1%milk in PBS were added to the plate. Subsequently, 10 μL of B cellsupernatant was added to each well. After an hour at room temperature,the plates were again washed 5 times with dH₂O. To each well was added50 μL of Rabbit anti-Human Fc-HRP with minimum anti-rabbitcross-reactivity (Jackson Laboratories; 1:8000 dilution). After 1 hourat room temperature, the plates were again washed 5 times with dH₂O and50 μL of TMB substrate (Neogen) were added to each well. The reactionwas stopped after 30 minutes by the addition of 50 μL of 1 Nhydrochloric acid to each well and the plates were read at wavelength450 nm.

Representative data resulting from the primary screen is shown below inTable 3. Positive wells were identified as those that were found to havea signal at least three times that of a control well. A total of 968positive antigen-specific B cell wells were identified in the primaryscreen. All of these wells were taken forward for screening in afunctional assay, as described below.

TABLE 3 Plate Well Primary O.D. 2357 G11 2.598 2361 G5 3.218 2372 B82.308 2383 H5 3.05 2398 C5 2.203 2401 G12 3.566 2413 G11 3.347 2384 G124.057 2388 A10 4.219 2407 G11 3.448IL-13-Induced Eotaxin Production Assay

All of the 968 ELISA positive wells were screened twice in anIL-13-induced Eotaxin-1 release assay. The assay was performed such thatonly wells containing a high concentration of antibodies or wellscontaining high affinity antibody were identified as neutralizing. Atotal of 78 neutralizing antibodies were identified as neutralizing inthis assay. The specific data from several wells of interest are alsoshown for illustrative purposes in Table 4.

For the assay, half of the area of 96-well assay plates was seeded with4000 HDFa cells/well in 50 μL of Medium 106 supplemented with low serumgrowth supplement (Cascade). The plates were then incubated overnight at37° C. in 5% CO₂ In a separate plate, 12.5 μL sample antibody, negativecontrol or positive control was aliquoted into sterile 96-well assayplates. Approximately 600 μM of IL-13 was prepared in Medium 106 (4×final concentration) and approximately 100 ng/mL TNF-alpha was preparedin Medium 106 (2× final concentration).

To begin the assay, 12.5 μL of IL-13 or media alone was added to eachwell and allowed to incubate 37° C. in 5% CO₂ for 1 hr. Following the 1hr incubation, the media of the HDFa cell was carefully removed using amultichannel pipette. 25 μL of TNF-alpha was added to each well. 25 μLsample/IL-13 was transferred to HDFa/TNF-alpha wells and cells wereincubated at 37° C. in 5% CO₂ for 48 hrs.

Following 48 hrs of incubation, supernatant from HFDa assay wells wascollected into 96-well VEE bottom plate. Samples were centrifuged at1500 rpm for 5 min.

30 μL of sample was assayed for Eotaxin-1 release in an assay kit (R&Dsystems) according to standard protocol with the followingmodifications. (1) 50 μL Capture Ab was coated at 2 μg/mL; (2) 50 μLsample or standard was used (30 μL sample+20 μL media for a final volumeof 50 μL); (3) 50 μL of detection Ab was used at 0.1 μg/mL; (4) 50 μLStreptavidin-HRP was used at 0.5 μg/mL; and (5) 50 μL Substrate Solutionwas used.

TABLE 4 Eotaxin Eotaxin ELISA Concentration % ELISA Concentration %Plate Well O.D. (pg/mL) Inhibition O.D. (pg/mL) Inhibition 2357 G110.429 25 79 0.283 13 80 2361 G05 0.393 19 85 0.295 15 76 2372 B08 0.53241 72 0.282 13 80 2383 H05 0.42 23 84 0.247 6 90 2398 C05 0.34 11 900.228 3 96 2401 G12 0.564 46 70 0.384 31 57 2413 G11 0.401 20 84 0.28313 82 2384 G12 0.517 38 73 0.297 15 76 2388 A10 0.459 29 78 0.274 11 822407 G11 0.469 31 78 0.278 12 84High Antigen (HA) Analysis of Anti-IL-13 Specific B Cell Culture Wells

Using an ELISA method, supernatants for concentration of antigenspecific antibody were normalized. Using an anti-target (IL-13) antibodyof known concentration titrated in parallel, a standard curve wasgenerated and the amount of antigen specific antibody in the supernatantwas compared to the standard and its concentration determined, see Table5 below.

TABLE 5 ELISA OD Ab Concentration determined at different (ng/ml) Basedon Plate Well antibody dilutions an anti-IL-13 Standard Curve 2357 G113.944 1.769 0.708 0.424 386 2361 G5 4.483 2.345 0.794 0.438 532 2372 B83.209 1.238 0.552 0.373 240 2383 H5 4.389 2.361 0.768 0.438 523 2398 C52.057 0.752 0.383 0.324 114 2401 G12 4.312 2.285 0.796 0.441 521 2413G11 3.977 1.783 0.648 0.415 360 2384 G12 4.639 3.132 1.072 0.528 8562388 A10 4.689 3.23 1.261 0.612 1049 2407 G11 4.891 2.9 1.072 0.537 824

The amount of antigen-specific antibody in each well was quantitated andplotted against the neutralization data for that well to identify thehighest potency wells (FIG. 1). The wells containing the highest potencyantibodies are those with the best inhibition with the lowestconcentration of antibody (upper left quadrant of the graph).

Limited Antigen (LA) Analysis of Anti-IL-13 Specific B Cell CultureWells

The limited antigen analysis is a method that affinity ranks theantigen-specific antibodies prepared in B-cell culture supernatantsrelative to all other antigen-specific antibodies. In the presence of avery low coating of antigen, only the highest affinity antibodies shouldbe able to bind to any detectable level at equilibrium. (See, e.g., PCTPublication WO03/048730A2, incorporated herein by reference).

Here, biotinylated IL-13 was bound to streptavidin plates at fourconcentrations (250 ng/mL; 125 ng/mL; 62 ng/mL; and 31 ng/mL) for 1 hourat room temperature on 96-well culture plates. Each plate was washed 5times with dH₂O and 45 μL of 1% milk in PBS with 0.05% sodium azide wasadded to the plate. This was followed by the addition of 5 μL of B cellsupernatant to each well. After 18 hours at room temperature on ashaker, the plates were again washed 5 times with dH₂O. To each well wasadded 50 μL of Gt anti-Human (Fc)-HRP at 1 μg/mL. After 1 hour at roomtemperature, the plates were again washed 5 times with dH₂O and 50 μL ofTMB substrate were added to each well. The reaction was stopped by theaddition of 50 μL of 1M phosphoric acid to each well and the plates wereread at wavelength 450 nm.

However, a number of wells including 2388A10 and 2357G11 were clearlysuperior as measured by OD at the lowest antigen coating, as illustratedin FIG. 2. The results presented in FIG. 2 demonstrate the ability ofthe different antibodies to bind at low concentration of antigencoating. The antibodies giving the highest OD signals have the highestaffinity under the conditions of this assay. The remaining clones werefurther analyzed by combining the high antigen data which measuresspecific antibody concentration and the limited antigen output. In thisway it was possible to compare the affinity of antibodies at differentconcentrations in B-cell culture supernatants. The wells containing thehighest affinity antibodies are those with the highest ELISA OD in thecontext of lowest concentration of Ag-specific antibody.

Based on all of the screening data, the wells listed in Table 6 wereidentified for further analysis (plaque assay and micromanipulation,single cell PCR and recombinant expression). Five wells were selectedbased on potency (inhibition/total specific Ab): 2372B8, 2398 C5,2401G12 and 2413G11. Three wells were selected based on affinity andinhibition: 2357G11, 2361G5 and 2384G12, and two wells were selectedbased on neutralization data alone: 2388A10 and 2407G11.

TABLE 6 ELISA OD determined at different antigen coatings Plate Well 250ng/ml 125 ng/ml 62 ng/ml 31 ng/ml 2357 G11 2.582 1.553 1.066 0.59 2361G5 2.582 1.505 1.075 0.423 2372 B8 1.616 0.79 0.506 0.234 2383 H5 1.5330.817 0.459 0.224 2398 C5 1.187 0.694 0.425 0.186 2401 G12 1.295 0.8270.407 0.198 2413 G11 1.274 0.783 0.449 0.203 2384 G12 2.056 1.161 0.7590.401 2388 A10 2.637 1.76 1.152 0.558 2407 G11 1.627 0.887 0.583 0.285IL-13-Specific Hemolytic Plaque Assay

Cells secreting IL-13-specific antibodies of interest were isolatedutilizing an IL-13 specific hemolytic plaque assay generally asdescribed in Babcook et al. (Proc. Natl. Acad. Sci. USA, 93:7843-7848(1996), incorporated herein by reference). The cells that were isolatedare identified in Table 7 below.

Biotinylation of Sheep Red Blood Cells (SRBC)

SRBC were stored in RPMI media as a 25% stock. A 250 μl SRBC packed-cellpellet was obtained by aliquoting 1.0 ml of the stock into an eppendorftube, spinning down the cells (pulse spin at 8000 rpm (6800 rcf) inmicrofuge) and removing the supernatant. The cells were then washedtwice with 1 ml of PBS pH 8.6. The cell pellet was then re-suspended in4.75 ml PBS at pH 8.6 in a 15 ml tube. In a separate 50 ml tube, 2.5 mgof Sulfo-NHS biotin was added to 45 ml of PBS at pH 8.6. Once the biotinhad completely dissolved, the 5 ml of SRBCs were added and the tuberotated at RT for 1 hour. The SRBCs were centrifuged at 3000 g for 5min, the supernatant drawn off and the SRBCs resuspended in 1 ml PBS atpH 7.4 in an Eppendorf tube. SRBCs were washed 3 times with 1 ml PBS atpH 7.4. The SRBCs were then resuspended in 4.75 ml immune cell media(RPMI 1640 with 10% FCS) in a 15 ml tube (5% B-SRBC stock). Stock wasstored at 4° C. until needed.

Streptavidin (SA) Coating of B-SRBC

One ml of the 5% B-SRBC stock was transferred into to a fresh eppendorftube. The B-SRBCs were pelleted, the supernatant drawn off, the pelletre-suspended in 1.0 ml PBS at pH 7.4, and the centrifugation repeated.The wash cycle was repeated 2 times, and then the B-SRBC pellet wasresuspended in 1.0 ml of PBS at pH 7.4 to give a final concentration of5% (v/v). 10 μL of a 10 mg/ml streptavidin (CalBiochem, San Diego,Calif.) stock solution was added and the tube mixed and rotated at RTfor 20 min. The washing steps were repeated and the SA-SRBC werere-suspended in 1 ml PBS pH 7.4 (5% (v/v)).

Human IL-13 Coating of SA-SRBC

The SA-SRBC were coated with photobiotinylated-Human IL-13-RbFc fusionat 100 ug/ml, then mixed and rotated at RT for 20 min. The SRBC werewashed twice with 1.0 ml of PBS at pH 7.4 as above. The IL-13-coatedSRBC were re-suspended in RPMI (+10% FCS) to a final concentration of 5%(v/v).

Determination of the Quality of IL-13-SRBC by Immunofluorescence (IF)

Approximately 10 μl of 5% SA-SRBC and 10 μl of 5% IL-13-coated SRBC wereeach added to separate fresh 1.5 ml eppendorf tube containing 40 μl ofPBS. A control human anti-IL-13 antibody was added to each sample ofSRBCs at 45 μg/ml. The tubes were rotated at RT for 20 min, and thecells were then washed three times with 100 ul of PBS. The cells werere-suspended in 50 μl of PBS and incubated with 20 μg/mL Gt-anti HumanIgG Fc antibody conjugated to Alexa488 (Molecular Probes, Eugene,Oreg.). The tubes were rotated at RT for 20 min, and then washed with100 μl PBS and the cells re-suspended in 10 μl PBS. 10 μl of the stainedcells were spotted onto a clean glass microscope slide, covered with aglass cover slip, observed under fluorescent light, and scored on anarbitrary scale of 0-4.

Preparation of Plasma Cells

The contents of a single B cell culture well previously identified bythe various assays described above as containing a B cell clonesecreting the immunoglobulin of interest were harvested. Using a100-1000 μL pipetteman, the contents of the well were recovered byadding 37C RPMI (+10% FCS). The cells were re-suspended by pipetting andthen transferred to a fresh 1.5 ml Eppendorf tube (final vol. approx700-1000 μl). The cells were centrifuged in a microfuge at 2500 rpm for1 minute at room temperature. The tube was then rotated 180 degrees andspun again for 1 minute at 2500 rpm. The freeze media was drawn off andthe immune cells resuspended in 100 μL RPMI (10% FCS), then centrifuged.This washing with RPMI (+10% FCS) was repeated and the cellsre-suspended in 75 μL RPMI (+10% FCS) and stored on ice until ready touse.

Plaque Assay

To a 75 μL sample of cells was added 75 uL each of IL-13-coated SRBC (5%(v/v) stock, diluted as necessary if the SRBC lawn was too thick), 4×guinea pig complement (Sigma, Oakville, ON) stock prepared in RPMI (+10%FCS), and 4× enhancing sera stock (1:900 in RPMI (+10% FCS)). Themixture (3-5 μL) was spotted onto TC plate lids (BD Biosciences, SanJose, Calif.) and the spots covered with undiluted paraffin oil. Theslides were incubated at 37° C. for a minimum of 1 hour.

TABLE 7 Single Cell (SC) Plate Well Numbers 2407 G11 SC-IL-13-557-5762388 A10 SC-IL-13-577-596 2401 G12 SC-IL-13-597-616 2372 B8SC-IL-13-617-636 2413 G11 SC-IL-13-637-657 2398 C5 SC-IL-13-658-670 2383H5 SC-IL-13-671-690 2384 G12 SC-IL-13-691-710 2357 G11 SC-IL-13-711-7302361 G5 SC-IL-13-731-750Cloning and Expression

After isolation of the single plasma cells, mRNA was extracted andreverse transcriptase PCR was conducted to generate cDNA encoding thevariable heavy and light chains of the antibody secreted by each cell.The human variable heavy chain region was cloned into an IgG2 expressionvector. This vector was generated by cloning the constant domain ofhuman IgG2 into the multiple cloning site of pcDNA3.1+/Hygro(Invitrogen, Burlington, ON). The human variable light chain region wascloned into an IgK or IgL expression vector. These vectors weregenerated by cloning the constant domain of human IgK or human IgL intothe multiple cloning site of pcDNA3.1+/Neo (Invitrogen, Burlington, ON).

The heavy chain and the light chain expression vectors were thenco-transfected using lipofectamine into a 60 mm dish of 70% confluenthuman embryonal kidney (HEK) 293 cells. The transfected cells secreted arecombinant antibody with the identical specificity as the originalplasma cell for 24-72 hours. 3 mL of supernatant was harvested from theHEK 293 cells and the secretion of an intact antibody was demonstratedwith a sandwich ELISA to specifically detect human IgG. Specificity wasconfirmed through binding of the recombinant antibody to IL-13 usingELISA.

The secretion ELISA tests were performed as follows. Control plates werecoated with 2 mg/mL goat anti-human IgG H+L overnight as for bindingplates, IL-13 was coated onto Costar Labcoat Universal BindingPolystyrene 96 well plates and held overnight at 4° C. The plates werewashed five times with dH₂O. Recombinant antibodies were titrated 1:2for 7 wells from the undiluted lipofection supernatant. The plates werewashed five times with dH₂O. A goat anti-human IgG Fc-specificHRP-conjugated antibody was added at a final concentration of 1 μg/mLfor 1 hour at RT for the secretion and the two binding assays. Theplates were washed five times with dH₂O. The plates were developed withthe addition of TMB for 30 minutes and the ELISA was stopped by theaddition of 1 M phosphoric acid. Each ELISA plate was analyzed todetermine the optical density of each well at 450 nm.

Purification of Recombinant Anti-IL-13 Antibodies

For larger scale production, heavy and light chain expression vectors(2.5 μg of each chain/dish) were lipofected into HEK293 cells in ten 100mm dishes that were 70% confluent. The transfected cells were incubatedat 37° C. for 4 days, at which time the supernatant (6 mL) was harvestedand replaced with 6 mL of fresh media. At day 7, the supernatant wasremoved and pooled with the initial harvest (120 mL total from 10plates).

Each antibody was purified from the supernatant using Protein-ASepharose (Amersham Biosciences, Piscataway, N.J.) affinitychromatography (1 mL). The antibodies were eluted from the Protein-Acolumn with 500 mL of 0.1 M Glycine (pH 2.5). The eluates were dialyzedin PBS (pH 7.4) and filter sterilized. The antibodies were analyzed bynon-reducing SDS-PAGE to assess purity and yield. Concentration was alsomeasured by UV analysis at OD 280.

Example 2 Recombinant Antibody Characterization

Recombinant antibodies were analyzed for potency in the eotaxin assay asdescribed above. The results are presented in Table 8 below. Alsoincluded are the measured IC₅₀'s in this assay for murine IL-13 receptorα2/FC and human IL-13 receptor α2/Fc. FIG. 3 shows the percentinhibition of IL-13 induced eotaxin release by recombinant antibodies643 and 731 compared to an isotype matched control, e.g., an irrelevantIgG2 monoclonal antibody.

TABLE 8 IC50 (pM) Standard mAb ID n = 1 n = 2 n = 3 Average Dev 731 1119 17 16 4 713 21 21 19 21 1 mIL-13Ralpha2/Fc 29 39 29 32 6 643 44 28 3335 8 623 31 40 35 36 4 693 38 69 53 54 16 602 80 53 ND 66 NA 353 99 12380 101 22 hIL-13Ralpha2/Fc 128 147 119 131 14 785 223 144 160 176 4211.18.3 213 304 217 245 51 157 260 207 306 258 50 176 233 ND ND 233 NA183 1040 1842 ND 1441 NA 264 293 313 284 297 15 243 253 ND ND 253 NA 3561087 913 ND 1000 NABiaCore Affinity

Affinity to human IL-13 (R&D) was investigated by BiaCore assay for sixof the antibodies (602, 623, 643, 693rep1, 693rep2 and 7310). First, twohigh-density goat α-human antibody surfaces were prepared on a CM5Biacore chip using routine amine coupling for the capture of the mAbsthree at a time. All mAbs were diluted to ˜5 μg/M1 using HBS-P runningbuffer containing 100 μg/ml BSA. Each purified mAb was captured for oneminute on a different flow cell surface for every IL-13 injection cycleusing a Biacore 2000 instrument.

IL-13 (R&D) was injected using the KINJECT command at concentrations of100.9, 50.4, 25.2, 12.6, 6.30, 3.15, 1.58 and 0.788 nM for mAbs 693, 713and 731 and 25.2, 12.6, 6.30, 3.15, 1.58, 0.788, and 0.394 nM for mAbs602, 623, and 643, over all surfaces for 1.5 min., followed by a twentyminute dissociation. The IL-13 samples were prepared in HBS-P runningbuffer containing 100 μg/ml BSA. All samples were randomly injected induplicate with several mAb capture/buffer KINJECT cycles interspersedfor double referencing.

The high-density goat α-human antibody surfaces were regenerated with a12-second pulse of 1/100 diluted concentrated phosphoric acid (146 mM,pH 1.5) after each cycle. mAb 693 was run twice because there was anextra flow cell available on the instrument during the last series ofmedium resolution experiments.

The data was fit to a 1:1 interaction model with a term for masstransport using CLAMP. The data for the six antibodies are shown inTable 9.

TABLE 9 Antibody k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (pM) 602 3.0 × 10⁶ 5.1× 10⁻⁴ 172 623 4.9 × 10⁶ 2.5 × 10⁻⁴ 52 643 4.4 × 10⁶ 2.9 × 10⁻⁴ 66 693rep 1 2.0 × 10⁶ 3.8 × 10⁻⁴ 189 693 rep 2 2.5 × 10⁶ 2.7 × 10⁻⁴ 109 7132.9 × 10⁶ 3.4 × 10⁻⁵ 12 731 3.9 × 10⁶ 3.5 × 10⁻⁵ 9Kinetic Analysis

Kinetic measurements of several of the antibodies were evaluated usingthe KinExA® method. This method involves solution-based determination offormal affinity measurements at equilibrium.

One hundred μg of each mAb was coupled to CNBr-activated Sepharose 4B orAzlactone beads. The remaining active groups on the beads were blockedas recommended by the manufacturer. The beads were then blocked with 10mg/ml BSA in 1 M Tris and stored in the blocking solution. For someexperiments the mAb was directly absorption coated to PMMA beads asrecommended by the manufacturer and blocked with 10 mg/ml BSA in PBS andstored in the blocking solution.

KinExA experiments were performed using an automated flow immunoassaysystem, KinExA 3000, in which beads coupled with the relevant mAbsserved as the solid phase. Briefly, a constant amount of native human ormacaque monkey IL-13 (10-650 pM), prepared by purifying and stimulatingPBMCs according to standard protocols, was incubated with titratingconcentrations of anti-h-IL-13 mAbs starting at 25 nM in sample buffer(PBS with 0.1% BSA to reduce nonspecific binding). Antigen/antibodycomplexes were incubated at RT for 48 hrs to 168 hrs to allowequilibrium to be reached. The mixture was drawn through thecorresponding antibody-coupled beads to accumulate unbound antigen. Thevolumes and flow rates of the mixture were varied depending upon thespecific signal obtained in each experiment.

The captured IL-13 was detected using solutions containing a secondaryAb (either a polyclonal anti-IL-13 Ab or a monoclonal Ab that binds toanother epitope) and Cy5-conjugated anti-species Ig to the secondaryantibody in sample buffer. In some cases the bead bound IL-13 wasdetected using a mixture of SA-Cy5 and a biotinylated antibody thatbinds to an epitope other than that bound by the bead immobilized Ab.

The concentrations, volumes, and flow rates of the secondary antibodysolutions were varied to optimize the signal to noise ratio in eachexperiment. The bound signals were converted into relative values as aproportion of control in the absence of hIL-13. Three replicates of eachsample were measured for all equilibrium experiments. The equilibriumdissociation constant (K_(D)) was obtained from nonlinear regressionanalysis of the data using a one-site homogeneous binding modelcontained within the KinExA software. The software calculates the K_(D)and determines the 95% confidence interval by fitting the data points toa theoretical K_(D) curve. The 95% confidence interval is given as K_(D)low and K_(D) high. The affinities are summarized in Tables 10 fornative human IL-13 and 11 for native macaque IL-13.

TABLE 10 Antibody K_(D) K_(D) low K_(D) high 623  24 pM 6.6 pM 60 pM 643 13 pM 6.2 pM 25 pM 713 3.6 pM 1.1 pM 7.3 pM  731 8.9 pM 6.2 pM 12 pM

TABLE 11 Antibody K_(D) K_(D) low K_(D) high 623  37 pM  18 pM  64 pM731 1.6 nM 880 pM 2.2 nM

The association rate constant was investigated using KinExA for two ofthe antibodies 623 and 731. The same IL-13 coupled beads were used asthe probe and the “direct” “injection” methods were used. These methodsare identical to the KinExA equilibrium assays with respect to antigencapture, antigen concentration and antigen detection. In the directmethod, the antigen and antibody are mixed in advance and then run onthe KinExA. In the injection method, the antibody and a titration ofantigen are mixed together for a set time before reading. Briefly,hIL-13 was mixed with an amount of mAb that would bind approximately 80%of the antigen based on the equilibrium experiments. The free antigenpresent in the sample was probed repeatedly, pre-equilibrium. Since thebinding signals are proportional to the concentration of free antigen inthe solution, the signals decreased over time until the solution reachedequilibrium. The volumes and flow rats of the antigen-mAb mixtures andthe Cy5-labeled secondary antibody were varied depending upon the mAbtested. Data was analyzed utilizing the KinExA analysis software. Thissoftware graphically represents the decrease in binding signals overtime, and fits the collected data points to an exact solution of thekinetic differential equations for binding. From this curve, an optimalsolution for the k_(on) was determined (Table 12). The k_(off) wasindirectly calculated from solutions for the k_(on) and K_(D).

TABLE 12 KD k_(on) bounds Antibody Method (pM) k_(on) (M⁻¹ · s⁻¹) k_(on)High k_(on) Low k_(off) (s⁻¹) % Error 623 Kinetic 24 1.1E+07 1.4E+075.1E+06 2.7E−04 1.37 Direct 623 Kinetic 24 1.5E+07 2.1E+07 1.1E+073.6E−04 5.46 inject 731 Kinetic 8.9 4.7E+06 6.3E+06 3.4E+06 4.2E−05 4.96injectBinding to the IL-13 Variant Protein

The ability of antibodies 623 and 731 to bind to an IL-13 variantprotein in which the wildtype arginine 110 is replaced with glutamine(IL-13Q110R) was investigated.

Briefly, plates were coated in IL-13RbFc (50 μL of 2.5 μg/mL) byincubation in 1×PBS (pH7.4) and 0.05% azide overnight at 4° C. Theplates were then washed with 1×PBS and blocked for 30 minutes with 100μL of 1% no fat skim milk/1×PBS at room temperature.

IL-13 or IL-13Q110R was pre-incubated with anti-IL-13 antibodies for 1hr at room temperature. Titrated IL-13 vertically from 2000 ng/ml withfinal volume of 30 μl/well. 30 μl of mAb was added per well at 40 ng/ml(sc731, 623) and 80 ng/ml (sc693), resulting in a final concentration ofIL-13 at the first point in the titration of 1000 ng/ml, a finalconcentration of antibodies 623 and 731 at the first point in thetitration of 20 ng/ml and final concentration of antibody 693 at thefirst point in the titration of 40 ng/ml.

After pre-incubation, 50 μl/well was transferred from the pre-incubationsolution to a plate pre-coated with IL-13RbFc and incubated for 30minutes at room temperature. Plates were washed and rabbit anti Hu IgGFc HRP was added at a concentration of 200 ng/ml. Following a further 30minutes incubation and subsequent wash, TMB was added and incubated foran additional 30 minutes. Reactions were stopped with 1N HCL and plateswere read as soon as possible on a Powerwave X340 96 well microplatereader (Biotek).

As can be seen in FIG. 4, pre-incubation with IL-13 inhibited binding ofboth antibodies 623 and 731 to IL-13 coated ELISA plates, whilepre-incubation with IL-13 variant IL-13Q110R inhibited binding of 731 toa much greater extent than binding of 623.

It is noted that antibodies to this particular variant can beparticularly useful in treating certain diseases. For example, as notedin U.S. Pat. Pub. No. 2005/0065327, a number of genetic polymorphisms inthe IL-13 gene have been linked to allergic disease. In particular, avariant of the IL-13 gene in which the arginine residue at amino acid130 is substituted with glutamine (Q130R) has been associated withbronchial asthma, atopic dermatitis and raised serum IgE levels (See,e.g., Heinzmann, A., et al. Hum Mol Genet, 2000. 9(4): p. 549-59;Howard, T. D., et al. Am J Hum Genet, 2002. 70(1): p. 230-6; Kauppi, P.,et al. Genomics, 2001. 77(1-2): p. 35-42; and Graves, P. E., et al. JAllergy Clin Immunol, 2000. 105(3): p. 506-13). This particular IL-13variant is referred to herein as the Q110R variant (arginine residue atamino acid 110 is substituted with glutamine) because a 20 amino acidsignal sequence has been removed from the amino acid count. Arima etal., (J Allergy Clin Immunol, 2002. 109(6): p. 980-7) report that thisvariant is associated with raised levels of IL-13 in serum. The IL-13variant (Q110R) and antibodies to this variant are discussed in WO01/62933. An IL-13 promoter polymorphism, which alters IL-13 production,has also been associated with allergic asthma (van der Pouw Kraan, T.C., et al. Genes Immun, 1999. 1(1): p. 61-5). It is believed that thevariant induces an increase in the incidence of asthma throughincreasing the effective half-life of the IL-13 ligand. It is believedthat IL-13Q110R can have a lower affinity for the decoy IL-13 receptor.

As will be appreciated by one of skill in the art, in light of thepresent disclosure, in some embodiments, the antibodies can bind to bothIL-13 and an IL-13 variant with approximately equal affinities orK_(D)s. This can allow one to treat patients with both forms of IL-13(wild-type and a variant) with a single antibody. Thus, in someembodiments, an antibody that can bind to IL-13 and an IL-13 variantwith a same or similar K_(D) can be useful. In some embodiments, thereis less than a 20% difference in the K_(D) of the fully human mAb forIL-13 and IL-13Q110R, for example, less than 20-15, 15-10, 10-8, 8-6,6-4, 4-2, 2-1, 1-0 percent difference in the K_(D)s of the antibody. Insome embodiments, the K_(D)s between the wild-type and IL-13Q110Rvariant differ by less than 1000 fold, 1000-100, 100-10, 10-1, or 1-0.2fold. Similarities for other variants of IL-13 can also be used inselecting or using antibodies.

Receptor Chain Competition

The ability of anti-IL-13 antibodies to block IL-13 binding to thereceptors IL-13Rα1 and IL-13Rα2 was investigated. Samples were analyzedusing the flow cytometer. The results are presented in FIG. 5A and FIG.5B. The data demonstrated the ability of Ab 643 (FIG. 5A) and of Ab 731(FIG. 5B) or an isotype control antibody to bind to IL-13 and thereceptors involved in the binding process. The particular receptor(e.g., IL-13Ra2, IL-13Ra1, or IL-4R) that was binding IL-13 and allowingthe antibody to interact with the cells was determined usingneutralizing antibodies against all possible IL-13 receptors expressedon HDFa cells. A summary of the various experiments and predictedresults is displayed in FIG. 5C and FIG. 5D.

Briefly, HDFa cells were resuspended in FACS buffer to yield about 200000 cells/well/100 μL and 100 μL of cells were aliquoted into 96-wellVEE bottom plates. Neutralizing anti-receptor antibodies (anti humanIL-13Ra1 (R&D Systems), anti human IL-13Ra2 (R&D Systems) or anti humanIL-4R(R&D Systems)) were diluted in FACS buffer at twice the finalconcentration (10 μg/mL FINAL). Anti-IL-13 and Control Ab's were alsodiluted in FACS buffer at 2× final concentration (1 μg/mL), as was IL-13(human R&D; 10 ng/mL FINAL).

A VEE bottom plate of HDFa cells was centrifuged at 180×g for 7 min andthe supernatant removed by inversion (PLATE #1). Cells were resuspendedin 50 μL FACS buffer and an additional 50 μL of anti human IL-13Ra1,anti human IL-13Ra2, anti human IL-4R or FACS buffer (No Receptor AbControl) was added to appropriate wells. The cells and antibodies werethen incubated on ICE for about 1.5 hrs.

A second VEE bottom plate was used for Ab/IL-13 pre-incubation (PLATE#2). 60 μL of the test antibody was aliquoted into a VEE bottom plate.60 μL of IL-13 added to appropriate wells and the mixture was incubatedon ice for about 1.5 hrs.

After the incubation HDFa cells were centrifuged at 180×g for 7 min andthe supernatant was removed by inversion. The cells in PLATE #1 wereresuspended in 100 μL FACS buffer or 100 μL of Ab/IL-13 and incubatedfor a further 1.5 hrs.

Following the second incubation the cells were centrifuged, washed 1×with FACS buffer and 100 μL of FACS buffer, 7AAD or 2 μg/mL goat anti HuIgG-Fc-Cy5 was added to appropriate wells.

The cells and secondary antibody were incubated on ice for 20 minutes,followed by a wash with FACS buffer. Cells were then resuspended in 100μL FACS buffer and aliquoted into pre-labeled FACS tubes containing 300μL cold FACS buffer.

Samples were analyzed using the flow cytometer. The results arepresented in FIG. 5A and FIG. 5B. A summary of the above protocol andpredicted results for each of the antibodies is shown in FIG. 5C andFIG. 5D. As shown by FIG. 5A, IL-13 does not bind to HDFa cells in thepresence of Ab 643. It appears that Ab 643 prevents IL-13 from bindingto its receptors on HDFa cells, as shown in each of the panels of FIG.5C. As can be seen in FIG. 5B, this is not the case for Ab 731. IL-13allows Ab731 to bind to HDFa cells. This binding is not blocked by Absagainst IL-13Ralpha1 or IL-4R but is blocked by antibodies againstIL-13Ralpha2, indicating that Ab 731 prevents IL-13 from binding toIL-13Ralpha1 or IL-4R but not to IL-13Ralpha2, as displayed in FIG. 5D.Results for mAb 623, an antibody that is similar to mAb 643, are alsopresented below.

The amount of IL-13 Ra1, IL-13 Ra2 and IL-4R surface expression on HDFacells was determined by FACS analysis using anti Receptor antibodies.HDFa cells prepared as described above were incubated with anti receptorantibodies at a concentration of 5 μg/ml on ice for 1 hr. Cells werewashed with FACS buffer and incubated with Cy5 secondary (anti-hum)antibody at 2 μg/ml. on ice for 30 min. After washing, samples wereanalyzed by flow cytometry. The results are presented in Table 13 below.

TABLE 13 FACS Geometric Antibody Target Mean Average IL13 Receptor Alpha1 8.39 IL13 Receptor Alpha 2 9.4 IL4 Receptor Alpha 1 9.15 NegativeControl 3.8

In addition to the above results for mAb 643 and mAb 731, a similarexperiment was performed for mAb 623. The results are shown in FIG. 5E.The results for mAb 623 are similar to those for mAb 643, suggesting aninteraction depicted in FIG. 5C, rather than that depicted for mAb 731in FIG. 5D.

Epitope Mapping

The epitopes for the antibody-IL-13 complexes were analyzed by threemethods, 1) SELDI, 2) Screening of Random peptide phage displaylibraries, and 3) expression of Chimeric Human/Mouse IL-13 molecules.These three techniques combined with knowledge of the structure of IL-13produced a coherent view of the relative binding sites and antigenicregions of these mAbs. This has permitted the identification offunctional epitopes, particularly for the regions involved in binding tothe signaling receptor.

As an initial examination, dot blot analysis of mAb binding to IL-13purified protein revealed which antibodies bound to which form (linearor conformational) of the epitope. mAbs 693 and 785 bound to the reduceddenatured antigen, the linear epitope. mAbs 602, 623, 643, and 713,bound to the non reduced (conformational epitope) IL-13 but not to thereduced denatured antigen. mAb 763 displayed no binding. Following this,the linear epitopes were mapped using random peptide phage displaylibrary. After two rounds of panning mAb 693 against a 12-mer randompeptide library expressed on phage, a single specific binder wassequenced and aligned to residues 109-120 (Helix D) of IL-13. (FIG. 6A).IL-13 antibodies were grouped in 3 different bins, although bins do notalways correlate with epitopes determined by other means. One antibodyfrom each bin was picked for mapping by SELDI. Table 14 demonstrates thebinning results of the IL-13 antibodies.

TABLE 14 Mab VH VL Bin 353 VH4-59/D2- A30/JK3 1 21/JH3b 713 VH3-23/D6-V2-1/JL1 1 19/JH6b 731 VH3-23/D6- V2-1/JL1 1 19/JH6b 602 VH3-15/D1-V2-7/JL3 2 26/JH6b 623 VH3-15/D1- V2-7/JL3 2 26/JH6b 643 VH3-15/D1-V2-7/JL3 2 26/JH6b 693 VH4-4/D5-5/JH6B V2-14/JL2 3Mapping of Epitopes Using SELDI

The antibody-antigen complex was digested with a high concentration ofLys-C and Asp-N. The epitope was then determined by SELDI and identifiedby the mass of the fragment. Table 15 displays the predicted masses forthe peptides derived by digestion of IL-13 digested with endoproteinaseLys-C.

TABLE 15 SEQ Mis. ID Mass Position Cut IL-13 Peptide Sequence NO: 9442.721–108 3 GPVPPSTALRELIEELVNIT 73 QNQKAPLCNGSMVWSINLTA GMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFS SLHVRDTK 7829.9 21–93 2GPVPPSTALRELIEELVNIT 74 QNQKAPLCNGSMVWSINLTA GMYCAALESLINVSGCSAIEKTQRMLSGFCPHK 7729.8 45–116 3 APLCNGSMVWSINLTAGMYC 75AALESLINVSGCSAIEKTQR MLSGFCPHKVSAGQFSSLHV RDTKIEVAQFVK 6815.3 45–108 2APLCNGSMVWSINLTAGMYC 76 AALESLINVSGCSAIEKTQR MLSGFCPHKVSAGQFSSLHV RDTK

The masses identified following cleavage were 6842.8 (for peptidefragment 45-108), 7733.7 (for peptide fragment 45-116), and 9461.4 (forpeptide fragment 21-108). Thus, the binding site for mAb 713 wasdetermined to be within residues 45-108 of IL-13.

Peptide Array for Mapping Conformational Epitopes

A peptide array of 101, 12-mer peptides, spanning residues 21-132 of theIL-13 sequence was generated (SIGMA-Genosys). Each consecutive peptidewas offset by one amino acid from the previous one, yielding a nested,overlapping library. The array was probed with mAb 713 and binding ofmAb 713 to the peptides was detected by incubating the PVDF membraneswith HRP-conjugated secondary antibody followed by enhancedchemiluminescence. Two consecutive spots, corresponding to amino acids70 to 80 of IL-13 and three consecutive spots, corresponding to aminoacids 83 to 92 of IL-13 were observed. The array was also probed withmAb 731. One spot, corresponding to amino acids 70 to 80 of IL-13 wasobserved. A similar experiment was also performed to determine theepitope for mAb 623, and is described in Example 10 below. The resultsindicated that mAb 623 binds to residues 21-29.

Epitope Mapping Using Mouse IL-13 Chimeric Molecules

Mouse sequences of Helix A, Helix B, Helix C, and Helix D were shuffledwith human sequences generating four new mouse chimeras. Arepresentation of the location of the helices is shown in FIG. 6B. Noneof the mAbs bound to the mouse IL-13. The four chimeras are as follows:

(SEQ ID NO: 77) MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPRSVSLPLTLKELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREG QFN; (SEQ ID NO: 78)MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGGFCVALDSLTNVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN; (SEQ ID NO: 79)MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIYRTQRILHGLCPHKVSAGQFSSLHVRDTKIEVAQFVKDLLLHLKKLFREGQFN; (SEQ ID NO: 80)MHPLLNPLLLALGLMALLLTTVIALTCLGGFASPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAHFITKLLSYTKQLFRHGQQFN.

The chimeras were then expressed and secreted IL-13 chimeric proteinswere detected in an ELISA assay. The results are summarized in Table 16,the “*” denotes that the binding was weak in the sandwich ELISA.

TABLE 16 Hu IL- Mo Mo Mo Mo mAb 13 HelixA HelixB HelixC HelixD EpitopeBin 693 Yes Yes Yes Yes No HelixD 3 785 Yes Yes Yes Yes No HelixD  713*Yes Yes Yes No Yes HelixC 1  731* Yes Yes Yes No Yes HelixC 1 602 YesYes Yes Yes Yes 2 623 Yes Yes Yes Yes Yes 2 643 Yes Yes Yes Yes Yes 2

The results of the above three studies of the epitopes of IL-13 aresummarized in Table 17.1.

TABLE 17.1 Phage mAb Display SELDI Spots Chimera Bin 3.1.2.4 21-33HelixA 693 109-121 HelixD 3 785 111-128 HelixD 713 45-108 70-80 and83-92 HelixC 1 731 70-80 HelixC 1 602 2 623 21-29 2 643 2

Thus, it appears that a number of different possible epitope positionsare used by the various antibodies disclosed herein.

Antibody Binning Analysis

Anti-IL-13 antibodies were grouped in three different bins by measuringthe ability of two antibodies to bind to antigen at the same time (oneantibody capturing the antigen on a bead and the other antibody used fordetection). The signal on the beads in the absence of antigen wassubtracted from the signal obtained in the presence of antigen. Thesignal of each detection antibody was divided by the signal of thecapture antibody to determine the fold increase in binding as shown inTable 17.2. The antibodies were then binned based on similar bindingpatterns on the capture antibodies. The data identified the presence ofthree bins of antibody binding for the nine detection antibodies tested(Table 17.2).

TABLE 17.2 Ab used for Detection 353 11.18 713 731 693 623 602 643 785G2 Ab on 353 1.0 2.3 0.6 0.6 2.5 3.1 2.9 3.0 3.3 0.2 Beads 11.18 4.2 1.06.6 6.9 2.3 0.9 0.8 0.9 0.7 0.5 BIN: 1 2 1 1 3 2 2 2 2 N/A

Briefly, mouse anti-human IgG1, 2, 3, 4 (BD Pharmingen 555784)conjugated beads were added to capture antibody (353 & 11.18; 5 ug/mL)in individual darkened eppendorf tubes. The tubes were rotated in thedark at 4° overnight. Beads were aliquoted to each well of a filterplate (2500 of each bead/well) and washed.

IL-13-RbIg (5 μg/ml) and controls (media only) were added to the filterplate 60 μl/well, which was then incubated in the dark at roomtemperature for 1 hour on a shaker and subsequently washed 2 times.

Secondary antibodies diluted in media at 60 μl/well (1 antibody perwell) were added. The antibodies were used at the followingconcentrations (353B-5 g/ml; 11.18.31-5 μg/ml; 713-0.56 μg/ml; 731-1.28μg/ml; 693-2.7 μg/ml; 623-5.7 μg/ml; 602-11 g/ml; 643-4.3 μg/ml; 785-5.5μg/ml; 763-5.7 μg/ml; G2 control-5 μg/ml). Plates were then incubatedfor two hours at room temperature and washed.

Biotinylated Mo-anti-Hulg G1, 2, 3, 4 (BD Pharmingen # 555785) dilutedin medium at 5 μg/ml was added to each well (60 μl/well) and the plateswere incubated in the dark for 1 hour on a shaker at room temperature.After washing 60 μl/well Streptavidin-PE (5 ug/mL; Pharm # 554061)diluted in medium was added. Plates were incubated in the dark for 20min on the shaker at room temperature and washed 2 times.

Each well was resuspended in 80 μl storage/blocking buffer (PBS, 10mg/ml BSA, 0.05% w/v sodium azide) by carefully pipette up and downseveral times to resuspend beads. Each well was analyzed by reading onLuminex with the gate set between 8,400 and 14,500.

The Luminex platform is a fluorescence bead based technology whichenables one to run multiple assays at once. The Luminex reader is ableto ascertain positive signaling events on different coded microspheres.This allows one to coat each bead separately, then mix thedifferentially coated microspheres together and then in one step assayantibody binding to each of the different microspheres. For isotypingantibodies, microspheres were coated in such a manner in that each beadwas able to specifically bind a particular heavy chain or light chainisotype. The microspheres were then mixed together and hybridomasupernatant for each antibody was added. After a 20 minute incubation,the microspheres were washed, and the bound antibody was detected usinga fluorescently labeled secondary antibody. The microspheres were thenread using the Luminex reader.

Example 3 In Vivo Data

Humanized IL-13 Mice

Humanized IL-13 mice, in which the gene encoding murine IL-13 wasdisrupted by the insertion of a cDNA encoding human IL-13, weregenerated at Lexicon (The Woodlands, Tex.). Mice were backcrossed ontothe A/J strain to ensure that the mice were susceptible toallergen-induced airway hyper-reactivity as previously described (Ewertet al., (2000) Am. J. Respir. Cell. Mol. Biol.).

To demonstrate that humanized IL-13 mice produce only human IL-13 and nomurine IL-13, cytokine production from OVA-specific CD4⁺ T cells derivedfrom humanized IL-13 mice (6-8 wk of age) were compared with CD4⁺ Tcells derived from WT mice. Mice were sensitized by i.p. injection with50 μg OVA/1 mg Imject Alum (Pierce, Rockford, Ill.) in 0.9% sterilesaline or with PBS (3 mice per treatment). Seven days aftersensitization, mice were sacrificed, and single-cell suspensions of thespleens were prepared. Erythrocytes were lysed, and the washedsplenocytes were resuspended at 5×10⁶ cells/ml in complete mediumconsisting of HL-1 (BioWhittaker, Walkersville, Md.) with 10%heat-inactivated FCS, 2 mM L-glutamine, and 50 mg/L neomycin sulfate.Splenocytes were then cultured for 4 days at 37° C. in the presence of200 μg/ml OVA to generate Ag-reactive CD4⁺ T cells. CD4⁺ T cells (5×10⁵cells/well) were isolated and then incubated with freshly isolatedmitomycin C (25 μg/ml)-treated splenocytes (5×10⁵ cells/well) from WTmice in complete medium in the presence of 200 μg/ml OVA in 96-wellplates (250 μl/well) for 96 hours.

Cell-free culture supernatants were collected and tested for cytokineproduction. Human and murine IL-13 (DuoSet, R&D Systems, Minneapolis,Minn.) concentrations were determined by ELISA according to themanufacturer's protocol. As expected, CD4⁺ T cells derived fromhumanized IL-13 mice after in vitro OVA restimulation produced humanIL-13 and no murine IL-13 (FIG. 7A). In contrast, CD4⁺ T cells derivedfrom WT mice produced murine IL-13 and no murine IL-13 (FIG. 7 B).

Airway Hyper-Reactivity

The anti-IL-13 antibodies 731 and 623 were tested in OVA-induced asthmamodels using the humanized IL-13 mice described above. For themeasurement of airway reactivity to the intravenous administration ofacetylcholine, a 24 day protocol was used. Briefly, mice were immunizedby an intraperitoneal injection of OVA (10 μg; crude grade IV; Sigma) inPBS (0.2 ml). PBS alone was used as a control. Fourteen days afterimmunization, mice were anesthetized with a mixture of ketamine andxylazine [45 and 8 mg per kilogram of body weight (mg/kg), respectively]and challenged intratracheally with 50 μl of a 1.5% solution of OVA oran equivalent volume of PBS as a control.

Seven days after the first antigen challenge, mice were challenged againintratracheally with either OVA or PBS. The 731 and 623 antibodies wereadministered intraperitoneally at a dose of 100 μg/mouse one day beforeeach challenge (days 13 and 20). Control mice received PBS or anirrelevant IgG2 as isotype control. Three days after the finalintratracheal challenge, mice were anesthetized with sodiumpentobarbital (90 mg/kg), intubated, ventilated at a rate of 120breaths/min with a constant tidal volume of air (0.2 ml), and paralyzedwith decamethonium bromide (25 mg/kg). After a stable airway pressurewas established, acetylcholine was injected intravenously (50 μg/kg),and the dynamic airway pressure was measured for 5 min. The airwayhyperresponsiveness (AHR) to the acetylcholine challenge was measured.The airway hyperresponsiveness to acetylcholine challenge is defined bythe time-integrated rise in peak airway pressure [airway-pressure-timeindex (APTI) in centimeters of H₂O×seconds]. * P<0.05, compared to theOVA+IgG2 control group [one-way analysis of variance (ANOVA) followed byFisher's least significant difference test for multiple comparisons].Treatment with 731 or 623 resulted in a complete reversal of OVA-inducedAHR (FIG. 8). In this example, complete reversal means that the additionof the antibody with OVA results in an effect similar to one in whichthere is no OVA and only antibodies are added (e.g., IgG2). n=4mice/group in the PBS, PBS+IgG2, PBS+731 and OVA groups; n=5 mice/groupin the OVA+IgG2 group; n=6 mice/group in the PBS+623 and OVA+731 group;n=8 mice/group in the OVA+623 group. Data are mean±SE.

OVA-Induced Mucus Production

An 18 day protocol was used for the measurement of OVA-induced mucusproduction. After subcutaneous priming with Ovalbumin (OVA, 25 μg; crudegrade IV) (Sigma) in 2 mg Imject Alum on days 0 and 7, mice wereanesthetized with isofluorane and challenged intranasally with 50 μl ofa 1.5% solution of OVA in PBS on days 14, 15, and 17. Control micereceived alum as priming or PBS as challenge.

The 731 and 623 antibodies were administered intraperitoneally at a doseof 100 μg/mouse on days 13, 15, and 17. Control mice received PBS. Onday 18 mice were sacrificed and lungs were collected after beingperfused. Lung tissue, including central and peripheral airways, wasfixed in 10% formalin, washed in 70% ethanol, dehydrated, embedded inglycol methacrylate, cut into 4-μM sections, mounted on slides, andstained with hematoxylin and eosin, plus Periodic acid-Schiff (PAS).Lung sections (one section per animal) were examined at 20×magnification. Five fields were selected randomly and for each sectionthe number of bronchi was counted in each field. Sections were scored ona scale from 0 to 4 (0: <5% PAS⁺ goblet cells; 1: 5 to 25%; 2: 25 to50%; 3: 50 to 75%; 4: >75%). To obtain the histologic goblet cell score(expressed as arbitrary units; U) the sum of the airway scores from eachlung was divided by the number of bronchi examined. Five out of eightmice died in the OVA treated group. No mice died in the other groups.Administration of 731 and 623 effectively reversed OVA-induced increasein mucus-containing cells in the airways (FIG. 9) Data are mean±SE. n=3for OVA/OVA/PBS group (initially n=8); n=8 for OVA/OVA/731 group, n=4for OVA/OVA/623 group; n=4 for OVA/PBS/PBS group, n=5 for Alum/OVA/PBS,and Alum/PBS/PBS groups. *p<0.01 vs OVA/OVA/PBS group by unpairedStudent t-test.

The above use of the murine models and mucus and AHR measurements fortesting asthma is an accurate and scientifically accepted model fortesting for the effectiveness of a drug for treating asthma.(Willis-Karp M., Murine models of asthma in understanding dysregulationin human asthma, Immunopharmacology, 25:48:263-8 (2000)). Moreover, themodel is predicted to be reliable for those IL-13 related disorders thatshare symptoms that are similar to at least one of the symptoms shown inthese mouse models. As such, this and similar such animal models will besufficient for similarly testing the other identified IL-13 relateddisorders.

Example 4 Structural Analysis of Antibodies

The variable heavy chains and the variable light chains for theantibodies shown in Table 1 were sequenced to determine their DNAsequences. The complete sequence information for all anti-IL-13antibodies are shown in the sequence listing submitted herewith,including nucleotide and amino acid sequences.

Table 18 shows the amino acid sequences of the heavy chain genes for avariety of the IL-13 antibodies described herein. Table 18 also showsthe amino acid sequences corresponding to the CDRs and framework regionsfor each antibody, along with a comparison to its germline sequence.

Table 19 shows the amino acid sequences of the kappa light chain genesfor a variety of the IL-13 antibodies described herein. Table 19 alsoshows the amino acid sequences corresponding to the CDRs and frameworkregions for each antibody, along with a comparison to its germlinesequence.

Table 20 shows the amino acid sequences of the lambda light chain genesfor a variety of the IL-13 antibodies described herein. Table 20 alsoshows the amino acid sequences corresponding to the CDRs and frameworkregions for each antibody, along with a comparison to its germlinesequence.

TABLE 18 SEQ Single ID Cell NO V Heavy/D/J FR1 CDR1 FR2 — 81 GermlineEVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS 157 22VH3-21/D1-26/JH3b EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS183 30 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS 176 26EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKCLEWVS 243 18EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKCLEWVS 264 14EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS 82 GermlineQVQLQESGPGLVKPSETLSLTCTVS GGSISSYYWS WIRQPPGKGLEWIG 353 6VH4-59/D2-21/JH3B QVQLQESGPGLVKPSETLSLTCTVS GGSISTYYWS WIRQPPGKGLEWIG —83 Germline EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS 713 34VH3-23/D6-19/JH6B EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS731 38 EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS — 84 GermlineEVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS 785 58VH3-23/D3-3/JH4B EVQLLESGGGLVQPGGSLRLSCAAS GFTFSSYAMS WVRQAPGKGLEWVS —85 Germline QVQLQESGPGLVKPSETLSLTCTVS GGSTSSYYWS WIRQPAGKGLEWIG 693 42VH4-4/D5-5/JH4B QVQLQESGPGLVKPSETLSLTCSVS GGSISSYYWS WIRQPAGKGLEWIG — 86Germline EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAWMS WVRQAPGKGLEWVG 623 50VH3-15/D1-26/JH6B EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAWMS WVRQAPGKGLEWVG643 46 EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAWMS WVRQAPGKGLEWVG 602 54EVQLVESGGGLVKPGGSLRLSCAAS GFTFSNAWMS WVRQAPGKGLEWVG — 87 GermlineQVQLVESGGGVVQPGRSLRLSCAAS GFTFSSYGMH WVRQAPGKGLEWVA 11.18 2VH3-33/D6-19/JH5B QVQLVESGGGVVQPGRSLRLSCVAS GFTFSSYDMH WVRQAPGKGLEWVA —88 Germline EVQLVESGGGLVKPGGSLRLSCAAS GFTFSSYSMN WVRQAPGKGLEWVS 356 10VH3-21/NA/JH6B EVQLVESGGGLVKPGGSLRLSCAAS GFTFSDYNMH WVRQAPGKGLEWVS SEQSingle ID Cell NO CDR2 FR3 CDR3 FR4 — 81 SISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR WGQGTMVTVSS 157 22 YISTSYNYIYYADSVKGRFTISRDNAKNSLYLQMNSLPAEDTAVYYCAR DQVGATLDAFDI WGQGTMVTVSS 183 30YISSSYNYIYYGDSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DQVGATLDAFDIWGQGTMVTVSS 176 26 YTSTSNSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDQVGATLDAFDI WGQGTMVTVSS 243 18 YISTSNSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DQVGATLDAFDI WGQGTMVTVSS 264 14YISTSNSYIYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR DQVGATLDAFDIWGQGTMVTVSS 82 YIYYSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCARWGQGTMVTVSS 353 6 YIYYSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCARDGGHYWDDAFDI WGQGTMVTVSS — 83 AISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK WGQGTTVTVSS 713 34 AFSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCVQ DGLGPYFYNYGMDV WGQGTTVTVSS 731 38AFSGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCVQ DGLGPYFYNYGMDVWGQGTTVTVSS — 84 AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWGQGTLVTVSS 785 58 AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKADFWSGTLWGFDY WGQGTLVTVSS — 85 RIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCAR WGQGTLVTVSS 693 42 RIYMTGRTNYNSSLKSRVTMSIDTSKNQLSLKLSFMTAADTAVYYCAR ESGSSYSYDY WGQGTLVTVSS — 86RIKSKTDGGTTDYAAPVKG RFTTSRDDSKNTLYLQMNSLKTEDTAVYYCTT WGQGTLVTVSS 623 50RIRSEIDGGTTNYAAPVKG RFTISRDDSKNTLYLQMNSLKTEDTAVYYCAT DQVGAYYGDYYGMDVWGQGTLVTVSS 643 46 RIRSEIDGGTTNYAAPVKG RFTISRDDSKNTLYLQMNSLRTEDTAVYYCATDQVGAYYGDYYGMDV WGQGTLVTVSS 602 54 RIRSKTDGGTINYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCAT DQVGAYYGDYYGMDV WGQGTLVTVSS — 87VTWYDGSNKYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR WGQGTLVTVSS 11.18 2VIWYDGSNKYYADSVQG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTS EDSSGWYDGWFDPWGQGTLVTVSS — 88 SISSSSSYIYYADSVKG RFTISRDNAKNSLYLQMNSLPAEDTAVYYCARWGQGTTVTVSS 356 10 STSYSSTYIYYADSVRG RFTISRDNAKNSLYLQMNSLRAEDTAVFYCAREDYYYYGLDV WGQGTTVTVSS

TABLE 19 SEQ Single ID Light--V Cell NO Kappa/J FR1 CDR1 FR2 89 GermlineDIQMTQSPSSLSASVGDRVTITC RASQGIRNDLG WYQQKPGKAPKRLIY 157 24 A30(Vk1)/JK3DIQMTQSPSSLSASVGDRVTITC RASQGIGDDLG WYQQKPGKAPKRLIY 183 32DIQMTQSPSSLSASVGDRVTITC RASQGIGDDLG WYQQKPGKAPKRLIY 176 28DIQMTQSPSSLSASVGDRVTFTC RASQDITDDLG WYQQKPGKAPKRLIY 243 20DIQMTQSPSSLSASVGDRVTFTC RASQDITDDLG WYQQKPGKAPKRLIY 264 16DIQMTQSPSSLSASVGDRVTFTC RASQDITDDLG WYQQKPGKAPKRLIY 353 8DIQMTQSPSSLSASVGDRVTITC RASQGIRNDLD WYQQKPGKAPKRLIY — 90 GermlineDIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WYQQKPGKVPKLLIY 11.18 4 A20/JK3DIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WYQQKPGKVPKVLIY — 91 GermlineDIQMTQSPSSLSASVGDRVTITC RASQGTRNDLG WYQQKPGKAPKRLIY 356 12 A20/JK2DIQMTQSPSSLSASVGDRVTTTC RASQGIRNDLG WYQQKPGKAPKRLIY SEQ Single ID CellNO CDR2 FR3 CDR3 J 89 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYCFGPGTKVDIK 157 24 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPFTFGPGTRVDIK 183 32 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPFTFGPGTKVDIK 176 28 AASSLQS GVPPRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPFTFGPGTKVDIR 243 20 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPFTFGPGTKVDIR 264 16 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPFTFGPGTKVDIR 353 8 DASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHDSYPFTFGPGTKVDIK — 90 AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC FGPGTKVDIK11.18 4 AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QKYNSAPFT FGPGTKVDIK —91 AASSLQS GVPSRFSGSGSGTEFTLTISSLQPEDPATYYC FGQGTKLEIK 356 12 AASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC LQHNSYPWT FGQGTKVEIK

TABLE 20 SEQ Single ID Light-V Cell NO Lambda/J FR1 CDR1 FR2 — 92Germline SYELTQPPSVSVSPGQTASITC SGDKLGDKYAC WYQQKPGQSPVLVIY 713 36V2-1/JL1 SYELTQPPSVSVSPGQTASITC SGDKLGDKYTC WFQQKPGQSPVLVIY 731 40SYELTQPPSVSVSPGQTASITC SGDKLGDKYAC WFQQKPGQSPVLVIY — 93 GermlineSYVLTQPPSVSVAPGQTARITC GGNNIGSKSVH WYQQKPGQAPVLVVY 693 44 V2-14/ JL2SYVLTQPPSVSVAPGQTARITC GGNNIGSKGVH WYQQKPGQAPVLVVY 785 60SYVLTQPPSVSVAPGQTARITC GGNNIGNKIVH WYQQKPGQAPVLVVY — 94 GermlineSYELTQPPSVSVSPGQTARITC SGDALPKKYAY WYQQKSGQAPVLVIY 623 52 V2-7/JL3SYELTQPPSVSVSPGQTARITC SGDALPEKYAY WYQQKSGQAPVLVIY 643 48SYELTQPPSVSVSPGQTARITC SGDALPEKYAY WYQQKSGQAPVLVIY 602 56SYELTQPPSVSVSPGQTARITC SGDALPEKYAY WYQQKSGQAPVLVIY SEQ Single ID Cell NOCDR2 FR3 CDR3 J — 92 QDSKRPS GIPERFSGSNSGNTATLTISGTQAMDEADYYC FGTGTKVTVL713 36 HDSKRPS GIPERFSGSNSGDTATLTISGTQAMDEADYYC QAWDSSTYV FGTGTKVTVL 73140 HDSKRPS GIPERFSGSNSGDTATLTISGTQAMDEADYYC QAWDSSTYV FGTGTKVTVL — 93DDSDRPS GIPERFSGSNSGNTATLTISRVEAGDEADYYC FGGGTKLTVL 693 44 DDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC QVWVSSSDHHVV FGGGTKLTVV 785 60 DDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYC QVWDSSSDHVV FGGGTKLTVL — 94 EDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYC FGGGTKLTVL 623 52 EDSKRPSGTPERFSGSSSGTMATLTTSGAQVEDEADYYC HSTDSSGNHGV FGGGTKLTVL 643 48 EDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYC HSTDSSGNHGV FGGGTKLTVL 602 56 EDTKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYC YSTDSSGNHGV FGGGTKLTVL

In some embodiments, the above sequences are used to generate variantsof the antibodies. For example, creating the variants can involve usingthe above sequences and structural breakdown (by sections or regions ofthe antibody) of the sequences to identify identical, similar, andnonconserved regions of the antibody. Sections of the antibody that arehighly conserved or identical will be maintained in the variant, whilesections that vary greatly between antibodies can be allowed to vary inthe new variant. Thus, “conserved” variants can readily be created byusing the above listing of antibody sequences. As will be appreciated byone of skill in the art, there are already many variants listed above,e.g., 713 and 731 or 623 and 643 which differ in their amino acidsequences. In some embodiments, no more than 40% of the amino acids ineach of the above sections (e.g., CDR1, CDR2, CDR3, FR1, J, etc.) areallowed to differ, for example, 40-30, 30-20, 20-15, 15-10, 10-5, 5-2,2-1, or 1% or less of the amino acids in each section are allowed to bechanged to nonconservative amino acids for the resulting antibody to beconsidered a variant antibody. As is demonstrated herein, the variantsappear to retain their various functions. Additional guidance can befound in identifying antibodies that bind to the same epitope and thenanalyzing the sequences for structural similarities (primary, secondary,tertiary, etc.) just between those antibodies.

In some embodiments, antibodies that bind to IL-13, with at least asubset of the above identified sequence are also contemplated. Forexample, an IL-13 fully human antibody with a CDR1 region as describedabove, or, more particularly, with a CDR1 region that has the sequenceGFTF in it can be used. Similarly, an antibody with a light chain CDR2region ending in Kn or a heavy chain CDR2 region ending in VKG, or aCDR3 region starting with GMDV can also be used. Any of the abovesequences or subsequences can similarly be used, especially when thesequences are common across antibodies.

As noted herein, in some embodiments, the antibodies bind with the sameaffinity to IL-13 as they do to other variants of IL-13. In someembodiments, the other variants of IL-13 include the Q110R variant. Suchantibodies, and variants thereof, can be created by the above disclosedmethods. Additional guidance can be found by sequence comparisons toidentify conserved regions in antibodies that bind with similar affinityto the wild-type and other IL-13 variants. IL-13 variants are known inthe art and are readily identified by one of skill in the art. Examplesof IL-13 variants can be found in Heinzmann et al., (Genentic variantsof IL-13 signaling and human asthma and atopy, Human Molecular Genetics,9:549-559 (2000)), incorporated by reference in its entirety.

Example 5 Use of Anti-IL-13 Antibodies as a Diagnostic Agents forDetection of IL-13 in a Sample

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of IL-13in a sample may be developed. In the assay, wells of a microtiter plate,such as a 96-well microtiter plate or a 384-well microtiter plate, areadsorbed for several hours with a first fully human monoclonal antibodydirected against IL-13. The immobilized antibody serves as a captureantibody for IL-13 that may be present in a test sample. The wells arerinsed and treated with a blocking agent such as milk protein or albuminto prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining IL-13, or with a solution containing a standard amount of theantigen.

After rinsing away the test sample or standard, the wells are treatedwith a second fully human monoclonal anti-IL-13 antibody that is labeledby conjugation with biotin. The labeled anti-IL-13 antibody serves as adetecting antibody. After rinsing away excess second antibody, the wellsare treated with avidin-conjugated horseradish peroxidase (HRP) and asuitable chromogenic substrate. The concentration of the antigen in thetest samples is determined by comparison with a standard curve developedfrom the standard samples.

Example 6 Treatment of COPD in Humans

A patient suffering from COPD is identified. A dosage of 5 mg/kg of theanti-IL-13 antibody 623 and/or 731 is administered by intravenousinjection to the patient. The level of eotaxin, C10, and/or TARC in thepatient is determined. If the level of eotaxin, C10, and/or TARC is toohigh, additional mAb is administered to the patient until a “normal”level of eotaxin, C10, and/or TARC is obtained. The anti-IL-13 antibodycauses an inhibition in the production of mucous, the development ofbronchial epithelium hyperplasia, and spasm of bronchial smooth muscle.This inhibition of mucous production and smooth muscle contractionreduces blockade of air passage with improved ventilation.

Example 7 Treatment of Chronic Bronchitis in Humans

A patient suffering from COPD characterized by chronic bronchitis isidentified. A dosage of 5 mg/kg of an anti-IL-13 antibody disclosedherein, preferably 623 or 731, is administered by intravenous injectionto the patient. The level of eotaxin, C10, and/or TARC in the patient isdetermined. If the level of eotaxin, C10, and/or TARC is too high,additional mAb is administered to the patient until a “normal” level ofeotaxin, C10, and/or TARC is obtained. The anti-IL-13 antibody causes apartial or complete inhibition of mucous production and bronchial smoothmuscle contraction in the inflamed respiratory tissues. This inhibitionof mucous production and smooth muscle contraction reduces blockade ofair passage with improved ventilation.

Example 8 Treatment of Emphysema in Humans

A patient suffering from emphysema is identified. A dosage of 5 mg/kg ofthe IL-13 antibody is administered by intravenous injection to thepatient. The level of eotaxin, C10, and/or TARC in the patient isdetermined. If the level of eotaxin, C10, and/or TARC is too high,additional mAb is administered to the patient until a “normal” level ofeotaxin, C10, and/or TARC is obtained. The anti-IL-13 antibody causes apartial or complete decrease in inflammatory cell infiltrate in therespiratory tissues. Additionally, the anti-IL-13 antibody may block theability IL-13 has to induce tissue damaging proteases.

Example 9 Treatment of Asthma in Humans

A patient suffering from asthma is identified. A dosage of 5 mg/kg of ananti-IL-13 antibody described herein, preferably 623 or 731, isadministered by intravenous injection to the patient. The level ofeotaxin, C10, and/or TARC in the patient is determined. If the level ofeotaxin, C10, and/or TARC is too high, additional mAb is administered tothe patient until a “normal” level of eotaxin, C10, and/or TARC isobtained. A booster administration is given later. The anti-IL-13antibody reduces the severity of tissue damage to the lungs and airpassages caused by the patient's immune response.

Example 10 Mapping of Conformational IL-13 Epitope Recognized by MAB 623

An overlapping peptide array (similar to that described in Example 2)spanning the human IL-13 sequence was generated to determine withgreater specificity where mAb 623 binds to IL-13. Because mapping wasinsufficient with standard procedures, an optimized protocol especiallysuited for the detection of conformational binding sites was used.Peptide scans containing 12-mer human IL-13-derived peptides shifted byone amino acid were probed with mAb 623. Binding of mAb 623 to thesearrayed human IL-13 derived peptides was analyzed by electrochemicaltransfer of the peptide bound antibody onto PVDF membranes, followed bydetection with a peroxides labeled anti-human IgG antibody andchemiluminescence. One binding region was identified. It appears thatthe epitope for mAb 623 for IL-13 includes TQNQKAPLCN (SEQ ID NO: 95)sequence (residues 20-29 in loop A, of SEQ ID NO: 96, FIG. 10).

Example 11 Human IL-13 High Resolution Screen with MAB 623

This example provides a further high-resolution assay and result for thebinding characteristics of mAb 623 to human IL-13. A goat anti-humanpolyclonal Ab (Fc specific) was amine coupled to all four flow cells ofa CM5 Biacore™ chip at high surface capacity (4800-5400 resonance units,RUs, of pAb) with a Biacore 2000™ instrument. The running buffer andsample preparation buffer for all experiments was degassed HBS-Pcontaining 100 μg/mL BSA. mAb 623 was diluted to 10.1 μg/mL in HBS-P forcapturing of the mAb on the biosensor surface. mAb 623 was captured onflow cells 1, 2, and 4. On average 552, 211, and 390 RUs of mAb werecaptured, respectively, on the three experimental flow cells for eachcycle using flow rates varying between 10-50 μL/min to achieve contacttimes varying between 12-60 seconds. Flow cell three served as acontrol. All IL-13 antigen (R&D Systems) injections were at 23° C. witha flow rate of 100 μL/min. Serially diluted (2-fold) IL-13 samples from12.6-0.394 nM were randomly injected in triplicate for 60 seconds withseveral buffer injections interspersed for double referencing. Thedissociation phase of the sensorgrams was followed for 30 minutes. Thecapture surface was regenerated with one, 15 second, injection of 146 mMphosphoric acid, pH 1.5. The sensorgram data were processed usingScrubber version 1.1 g. Data from all three flow cells were fit globallyto a 1:1 interaction model with a term for mass transport included; theR_(max) values for each flow cell were allowed to fit locally as isappropriate in this case since the capture levels were different on eachof the three flow cells. The model fit the data satisfactorily and gavethe results k_(a)=7.3×10⁶ M⁻¹s⁻¹, k_(d)=2.5×10 ⁻⁴ s⁻¹, K_(D)=34 pM.

Example 12 623 and 731 KINEXA Affinity for Marmoset IL-13

This example provides the affinity data for mAb 623 and mAb 731 formarmoset IL-13 from KINEXA analysis. K_(D) controlled as well as antigencontrolled experiments were done, similarly to the other KINEXAexperiments, for marmoset IL13 with mAb 623. The n-curve analysisrevealed that the final K_(D) was 403 pM. A number of experiments werealso performed for mAb 731 and by n-curve analysis, the K_(D) for 731was determined to be <7 pM.

Example 13 Medium Resolution Screen of MABS 623 & 731 with Human IL-13Variant (IL-13Q110R)

This example present the binding characteristics of mAb 623 and mAb 731to IL-13Q110R. Label-free surface plasmon resonance (SPR), or Biacore™device, was utilized to measure the antibody affinity to the antigen.For this purpose, a high-density goat α human antibody surface over aCM5 Biacore™ chip was prepared using routine amine coupling on a Biacore2000™ instrument. mAb 731 was diluted to 4.7 μg/mL and mAb 623 to 5.2μg/mL in degassed HBS-P (Hepes buffered saline containing 0.005%polysorbate 20) running buffer containing 100 μg/ml BSA. A captureprotocol was developed for both mAbs. Before each antigen sampleinjection, each mAb was captured over a different flow cell for 30seconds at a 10 μL/min flow rate. A, four minute, wash step at 100μL/min followed to stabilize the mAb baseline. Human IL-13 variant wasinjected (Peprotech, Inc.) for 90 seconds at a concentration range of23.6-0.185 nM (2× serial dilution) followed by a 15 minute dissociation.The IL-13 variant samples were prepared in HBS-P containing 100 μg/mL.All samples were randomly injected in triplicate with several bufferinjections interspersed for double referencing. The high-density goat αhuman antibody surfaces were regenerated with one 15 second pulse of 146mM phosphoric acid (pH 1.5) after each cycle. A flow rate of 100 μL/minwas used for all variant IL-13 injection cycles. The sensorgram datawere processed using Scrubber 1.1 g and were fit in Clamp 2000 to a 1:1interaction model with the inclusion of a term for mass transport. Theresulting binding constants are listed in Table 21. The data sets forboth mAbs were of high quality showing high reproducibility. A 1:1interaction model described both IL-13/mAb complexes adequately.

TABLE 21 Sample R_(max) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (pM) 731 455.11 × 10⁶ 5.02 × 10⁻⁵ 9.8 623 26 5.62 × 10⁶ 2.18 × 10⁻⁴ 38.8

Example 14 Inhibition of IL-13 and IL13Q110R Variant Induced EotaxinProduction

This example presents additional data regarding the ability of theantibodies to inhibit the variants of IL-13 and to inhibit eotaxinproduction. Anti-IL-13 antibodies were tested for their ability toinhibit eotaxin production in HDFa cells, a primary human dermalfibroblast cell line that expresses IL-13Rα1, IL-13Rα2 and IL-4Rα. Thecells were seeded overnight at 4000 cells/well in 96 well plates.Separately, IL-13 or IL-13Q110R was pre-incubated for an hour at 37° C.at 300 pM with or without anti-IL-13 antibodies at an initialconcentration of 10 nM. The IL-13 or IL-13Q110R and antibody mixture wasthen added to the cells treated with 50 ng/mL TNFα and incubated for 2days at 37° C. At this point the supernatants were collected andanalyzed for the presence of eotaxin using a quantitative ELISA. Theexperiments were conducted two or three times with triplicate datapoints. The results are presented in Table 22 below. Also included areIC₅₀'s for IL-13 measured in this assay. FIGS. 11A-D show the percentinhibition of the eotaxin release induced by IL-13 or IL-13Q110R variantby recombinant antibodies 623 and 731 compared to an isotype matchedcontrol, e.g., an IgG2 control monoclonal antibody.

TABLE 22 IC50 (pM) 300 pM IL- 600 pM IL- 600 pM IL- 300 pM IL-13 13Q110R13 13Q130R 623 56 106 113 277 731 28 54 56 140

Example 15 Neutralization of IL-13 Bioactivity In Vitro by 623 and 731

This example demonstrates the ability of the antibodies to inhibit ofHDLM-2 and L-1236 cell proliferation, two IL-13-responsive Hodgkin'slymphoma-derived cell lines. It has been shown that these cell lines notonly secrete IL-13 but also use this cytokine as a growth factor,possibly by an autocrine or paracrine mechanism (Trieu et al., Claudio JO et al., Soluble interleukin-13Ralpha2 decoy receptor inhibitsHodgkin's lymphoma growth in vitro and in vivo, Cancer Res, 64:3271-3275(2004)). After 72 hours of incubation with the relevant compound, cellproliferation was assessed by 3H-thymidine incorporation. Percent cellproliferation in the presence of inhibitor was calculated compared tocells alone control wells (100% production). Values were plotted asIL-13 inhibitor concentration (nM) vs percent cell proliferation. Datarepresent the average of five (L-1236 assay) and four (HDLM-2 assay)experiments. IL-13 neutralization by mAb 623 and mAb 731 resulted in adose-dependent inhibition of proliferation of both cell lines (FIG. 12Aand FIG. 12B). mAb 623 had EC₅₀s of 390 pM in the L-1236 proliferationassay (FIG. 12A) and 4.5 nM in the HDLM-2 proliferation assay (FIG.12B). 731 had EC₅₀s of 5.2 pM in the L-1236 proliferation assay (FIG.12A) and 0.18 nM in the HDLM-2 proliferation assay (FIG. 12B).hIL-13Rα2/Fc had EC₅₀s of 59 pM in the L-1236 proliferation assay (FIG.12A) and 0.6 nM in the HDLM-2 proliferation assay (FIG. 12B). The levelsof IL-13 measured in the supernatant of HDLM-2 and L-1236 cells were 2.6ng/ml and 118 pg/ml respectively.

Example 16 Inhibition of IL-13-Induced CD23 Expression in B Lymphocytes

IL-13 has been shown to induce CD23 up-regulation in B lymphocytes(Punnonen et al., Interleukin 13 induces interleukin 4-independent IgG4and IgE synthesis and CD23 expression by human B cells, Proc Natl AcadSci USA, 90:3730-3734 (1993)). This Example demonstrates mAb 623 and mAb731's ability to inhibit IL-13-induced expression of CD23 on Blymphocytes in whole blood. Increasing concentrations of mAb 623, mAb731 or isotype control were added to human whole blood in the presenceof recombinant human IL-13 (10 ng/ml). After 24 hrs at 37° C., B-cellswere immunostained using anti-CD19 and anti-CD23 antibodies and analyzedby FACS. Results (FIG. 13) were expressed as Geometric Mean expressionof surface CD23 on CD19⁺ cells compared to control wells containingIL-13 alone. Data represent the average of four (hIL-13Rα2/Fc), six(731) and eleven (623 and hIgG2) donors.

IL-13 neutralization by mAb 623 and mAb 731 resulted in a dose-dependentinhibition of CD23 expression with an IC₅₀ of 6.2 nM and 0.87 nMrespectively. hIL-13Rα2/Fc had an IC₅₀ of 4.0 nM (FIG. 13).

Example 17 Additional Testing of MAB 623 and 731 in Asthma Models

As noted above, since mAb 623 and mAb 731 do not cross-react with murineIL-13, humanized IL-13 mice were generated from 129×C57BL/6 mice by thegenetic disruption of the murine IL-13 gene through the introduction ofthe cDNA encoding the human IL-13 gene (Lexicon, The Woodland, Tex.).

Additional Ovalbumin (OVA)-Induced Asthma in IL-13 Humanized Mice:Prophylactic Studies

MAB 623 and MAB 731 Fixed Dose Prophylactic OVA Study

This experiment was performed in a manner similar to Example 3 above,using the 24 days protocol. The samples were examined for theeffectiveness of the antibodies on reducing OVA-induced mucus productionin the airways. Thus, the example demonstrates the effectiveness of theantibodies in treating asthma and similar IL-13 related disorders.

To examine the effect of mAb 623 and mAb 731 on mucus content of theairway epithelium, lungs slides were stained with hematoxylin and eosinplus Periodic acid-Schiff (PAS). Lung sections (one section per animal)were examined at 20×magnification. Five fields in each section wereselected randomly. In each field, the percentage of PAS-positive gobletcells in each brochus was counted. Data are expressed as the averagepercentage of PAS-positive goblet cells/bronchus. Data are mean±SE(shown in FIG. 14). In the control groups the number of goblet cellscontaining mucus per bronchus is less than 4%. OVA challenge increasedthe number of mucus-containing cells in the bronchi to 19% in the OVAgroup and to 37% in the OVA+IgG2 group. As can be observed in thefigure, treatment with mAb 623 reduced the percent of mucus-containingcells in the bronchi to 9%. Treatment with mAb 731 reduced the percentof mucus-containing cells in the bronchi to 2%.

To further examine the effect of mAB 623 and mAb 731 on the leukocyterecruitment in the airways, after the AHR measurements (Example 3), micewere sacrificed and bronchoalveolar lavage fluid (BALF) was collected byflushing the lungs two times with 1 ml of PBS. Cell counts weredetermined by light microscopic evaluation of cytospin preparations.Data are mean±SE and are shown in FIG. 15. Treatment with 623 or 731 hadno noticeable effect on OVA-induced leukocyte recruitment in BALF.

MAB 623 And MAB 731 Prophylactic Dose Response OVA.

This example demonstrates the dose dependency of the inhibitory effectof mAb 623 and mAb 731 on OVA-induced AHR, mucus production andleukocyte recruitment in the BALF. Mice were immunized according the 24day protocol described above in Example 3 (used for obtaining the datafor FIG. 8). On days 13 and 20 either mAb 623 or mAb 731 wereadministered ip at the doses of 0.3, 1, 3 or 10 mg/kg. Control micereceived PBS or an irrelevant IgG2 (10 mg/kg) as isotype control. APTIwas determined as described above (for FIG. 8). n=4 mice/group in thePBS, and OVA groups; n=6 mice/group in the PBS+IgG2, and OVA+IgG2groups; n=7 mice/group in the OVA+623 (3 mg/kg), and OVA+731 (0.3 mg/kg)groups; n=8 mice/group in the OVA+623 (10 mg/kg), OVA+623 (1 mg/kg),OVA+623 (0.3 mg/kg), OVA+731 (10 mg/kg), OVA+731 (3 mg/kg), and OVA+731(1 mg/kg) groups. Data are mean±SE. At the dose of 0.3 mg/kg neither 623nor 731 inhibited OVA-induced AHR, whereas doses of 1 mg/kg and higherinhibited AHR to acetylcholine to baseline (PBS) (see, FIG. 16).

This section of the example further demonstrates the dose dependency ofthe inhibitory effect of mAb 623 and mAb 731 on OVA-induced mucusproduction. Lungs were collected and treated as described above (forFIG. 14). Data are expressed as the average percentage of PAS-positivegoblet cells/bronchus. Data are mean±SE. Both mAb 623 and mAb 731 showeda similar dose dependent inhibition of the % of PAS+ cells in theairways of OVA treated mice (FIG. 17).

This section of the example further demonstrates that mAb 623 and mAb731 did not inhibit OVA-induced leukocyte recruitment in BALF at any ofthe doses tested (FIG. 18). BALF was collected and cell counts wereperformed as described above (in reference to FIG. 15). Data aremean±SE.

In this experiment the isotype control IgG2 tested at the dose of 10mg/kg inhibited OVA-induced AHR by 85%. On the other hand the isotypecontrol IgG2 had no effect on mucus hyperplasia and caused an apparentincrease in the number of leukocyte in BALF compared to the OVA group by146%. Thus, some variability in the effectiveness of the antibodies isto be expected and overcoming this variability will be routine in lightof the present teachings and the knowledge of one of skill in the art.The results indicate that elevated levels of the antibodies can berequired to observe the particular phenotype of inhibition ofOVA-induced leukocyte recruitment in BALF.

Example 18 House Dust Mite (HDM)-Induced Asthma in IL-13 Humanized Mice:Prophylactic and Therapeutic Studies

MAB 623 And MAB 731 Dose Response Prophylactic HDM Example.

The major allergen in house dust comes from mites. This Example uses aclinically relevant and representative allergen for an asthma model. Oneof skill in the art would consider this example and its results to berepresentative of the effectiveness of the antibodies in otherorganisms, including humans.

On days 1, 7 and 14 mice were challenged with three intratrachealadministrations of HDM (100 μg) in 50 μl of PBS. On days −1, 6 and 13either 623 or 731 were administered intraperitoneally at the doses of10, 3, 1, 0.3 mg/kg. Control mice received PBS or an irrelevant (e.g.,negative control) IgG2 as isotype control at the dose of 10 mg/kg. Onday 17 mice airway reactivity to the intravenous administration ofacetylcholine was measured as described above (FIG. 8). n=2 mice/groupin the PBS group; n=3 mice/group in the HDM, HDM+IgG2 groups; n=4mice/group in the PBS+IgG2 group; n=6 mice/group in the HDM+731 (10mg/kg), and HDM+731 (0.3 mg/kg) groups; n=8 mice/group in the HDM+623(10 mg/kg), HDM+623 (3 mg/kg), OVA+623 (1 mg/kg), HDM+623 (0.3 mg/kg),HDM+731 (3 mg/kg), and HDM+731 (1 mg/kg) groups. Data are mean±SE. Theresults are shown in FIG. 19. At the dose of 0.3 mg/kg 623 and 731 hadno effect on HDM-induced AHR, whereas doses of 1 mg/kg and higherinhibited AHR to baseline levels (FIG. 19). Due to the small number ofanimals in the HDM and HDM+IgG2 groups, AHR measured in these groups wasvery variable (APTI 579+463 and 415+213 in the HDM and HDM+IgG2 groupsrespectively). At the dose of 10 mg/kg mAb 623 reduced the percent ofPAS⁺ cells in the lung of mice challenged with HDM to baseline (PBSgroup).

In a similar fashion to that described above, dose responsiveness of theinhibition of HDM-induced mucus production by mAb 623 mAb 731 in IL-13humanized mice was examined. Lungs were collected and treated asdescribed in reference to FIG. 14 and were examined. Data are expressedas the average percentage of PAS-positive goblet cells/bronchus. Dataare mean±SE. At the dose of 10/mg/kg mAb 623 reduced the % of PAS+ cellsto baseline, whereas lower doses were not as effective. At the dose of0.3 mg/kg mAb 731 had no effect on the percent of PAS+ cells, but athigher doses it reduced the percent PAS+ cells to baseline levels (PBSgroup) (FIG. 20).

In a similar fashion to the experiments described above, mAb 623 and mAb731 were tested and determined to inhibit HDM-induced leukocyterecruitment in a dose dependent manner, starting at the dose of 1 mg/kg(see FIG. 21). BALF was collected and cell counts were performed asdescribed in relation to the data for FIG. 15. Data are mean±SE. Theisotype control caused an apparent increase in the number of leukocytesrecruited by HDM in the BALF (497±156 compared to 198±42 10³ cells/mlBALF in the HDM+IgG2 and HDM group respectively).

MAB 623 Fixed Dose Therapeutic and Prophylactic HDM Study.

This Example demonstrates the effectiveness of mAb 623 as a therapeuticand prophylactic in the HDM model. In this example, mAb 623 wasadministered at the fixed dose of 100 μg/mouse according to 3 differentschedules: i) one day before each HDM challenges (prophylactictreatment); ii) one day before the last HDM challenge and iii) at thesame day of the last HDM challenge (therapeutic treatments). Theallergic phenotype was assessed 3 days after the last HDM challenge.Thus, the timing of the administration of the antibody, and theresulting effectiveness, were determined. In alternative embodiments,this approach can be applied for the other antibodies.

Mice were challenged with three intratracheal administrations of HDM(100 μg) in 50 μl of PBS at day 1, 7 and 14. 623 or an IgG2 isotypecontrol were administered intraperitoneally at the dose of 100 μg/mouseaccording to 3 different schedules: on day −1, 6 and 13 (prophylactictreatment), on day 13 or on day 14 (therapeutic treatment). Control micereceived PBS or an irrelevant IgG2 as isotype control. On day 17 airwayreactivity to the intravenous administration of acetylcholine andleukocyte infiltration in BALF were measured as described above (inreference to FIG. 8 and FIG. 15). n=8 mice/group in the PBS, HDM,HDM+IgG2 (day −1, 6 and 13), HDM+IgG2 (day 13), HDM+IgG2 (day 14),HDM+623 (day 13), groups; n=10 in the HDM+623 (day −1, 6 and 13),HDM+623 (day 14) groups. Data are mean±SE. As shown in FIG. 22A, whenadministered prophylactically before each HDM challenge, mAb 623completely inhibited HDM-induced AHR. Furthermore, 623 completelyinhibited HDM-induced AHR when therapeutically administered the daybefore the last HDM challenge. In contrast, the therapeuticadministration on the same day of the last challenge had little effecton AHR (FIG. 22A). mAb 623 inhibited leukocyte recruitment in BALF whenadministered prophylactically before each HDM challenge (FIG. 22B).

As will be appreciated by one of skill in the art, the examples used forcharacterizing these antibodies can readily be applied to any antibodyto IL-13 and variant thereof. Thus, the examples represent how one ofskill in the art could readily and routinely determine whether anantibody, or a variant of an antibody disclosed herein, could functionin altering IL-13 activity.

As will be appreciated by one of skill in the art, while the aboveresults frequently focuse upon the prophylactic aspects of theantibodies, the above methods and results are extendable to methods fortreatment as a therapy. For example, once a subject has been identifiedthat is suffering from an IL-13 related disorder, an effective amount ofthe antibody can be administered. As will be appreciated by one of skillin the art, additional amounts of the antibody can be required comparedto the amount for a prophylactic use. For example, the amount inaddition can be 1-10, 10-100, 100-1000 fold or more above the amountsfor prevention, described above. This can be required where the disorderresults in large amounts of IL-13 or when large amounts of IL-13 have tobe removed in order to reduce the symptoms of the disorder. Inalternative situations, lower amounts of the antibody can be required totreat a subject with an IL-13 related disorder than to prevent thedisorder. For example, in situations in which a single dose of theantibody is sufficient to bind and remove substantially all of theexcess IL-13, whereas, to prevent the disorder, a similar amount, but anamount administered continuously, may have to be administered.Additionally, the amount can be administered in various ways, forexample an i.v., and the amount can be administered continuously, ifdesired. The above results are fully consistent with and suggestive ofthe fact that the antibodies will work as a therapeutic as well as aprophylactic. As will be appreciated by one of skill in the art,multiple and/or continuous doses, e.g., treatment for the life of thesubject, may be required in some situations.

Additionally, as will be appreciated by one of skill in the art, thenature of the particular host can also influence the manner and amountof treatment. For example, in subjects that are chronic (naturallysensitive to the compound and need not be sensitized to it) for adisorder (e.g., asthma) additional amounts, given by more efficientmeans (e.g. i.v. instead of s.c.) over a longer period of time would beexpected to have a higher likelihood of working, in light of the aboveresults and the knowledge of one of skill in the art. For example, whilethe above models and doses can be representative of acute models ofinfection, the above doses can be insufficient when administered asdescribed above, in one form of chronic model (e.g., in monkeys that hadbeen exposed to various agents over prolonged periods of time). Thisresult is consistent with what one of skill in the art would expect aselevated levels of IL-13 would logically require additional amounts ofthe antibody. Furthermore, one of skill in the art would expect that,given larger doses of the antibody, administered frequently enough, thatthe same or similar results, as described above, would result.Applicants note that this is consistent with the results shown in PCTpublication No. WO 2005/007699, which demonstrates that while low levelsof antibodies to IL-13 were not substantially effective in monkeymodels, that higher levels did result in significant and predictedresults. As noted above, one method by which the levels of antibodyrequired (for any IL-13 depend disorder) can be determined is throughthe monitoring of the levels of biomarkers in the subject. Examples ofthese biomarkers are described herein.

Example 19 Characterization of IL-13 Dependent Biomarkers: Effect of MAB623 ON Serum Levels of TARC, Eotaxin and C10 in the OVA-induced AsthmaModel in IL-13 Humanized Mice

This Example demonstrates and verifies serum IL-13-dependent biomarkersthat can be used in the clinical setting. The serum levels ofOVA-induced TARC, C10 and eotaxin were measured in mice and the effectof IL-13 inhibition on those levels were studied. (IL-13 has been shownto induce the release of TARC, C10, and eotaxin (see, e.g., Ma et al.,The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesisof IL-13-induced inflammation and remodeling, J Immunol., 172(3):1872-81(2004); Zhu et al., IL-13-induced chemokine responses in the lung: roleof CCR2 in the pathogenesis of IL-13-induced inflammation andremodeling, J Immunol. 168(6):2953-62 (2002); Nomura et al.,Interleukin-13 induces thymus and activation-regulated chemokine (CCL17)in human peripheral blood mononuclear cells, Cytokin,. 20(2):49-55(2002); Zhu et al., Pulmonary expression of interleukin-13 causesinflammation, mucus hypersecretion, subepithelial fibrosis, physiologicabnormalities, and eotaxin production, J Clin Invest., 103(6):779-88(1999))). As will be appreciated by one of skill in the art, thetechnique outlined in this approach could be used to identify othermarkers as well.

An OVA-induced asthma study in A/J wild type mice was first performed toestablish the time of serum induction of TARC, eotaxin and C10. On days1 and 7 mice were immunized with OVA (25 μg OVA in 2 mg Alum, ip) orAlum as control. On days 14, 15 and 17, mice were anesthetized with amixture of ketamine and xylazine [45 and 8 mg/Kg, respectively] andchallenged wit OVA (750 μg in 50 μL, intranasal) or an equivalent volumeof PBS as a control. Blood was collected 3, 6 and 24 hours after thefinal challenge. Serum levels of TARC and eotaxin were measured by ELISA(Duoset, R&D System). Serum levels of C10 were measured by sandwichELISA. Briefly, serum samples were titrated 1:2 on anti-C10 antibody(R&D System) coated plates for 1 hour. Biotinylated anti-C10 detectionantibody (R&D System) was added, followed by an incubation of 1 μg/mlstreptavidin-HRP. Captured C10 was determined using a TMB substratereaction and ng/ml values in each sample were quantitated from astandard curve on the plate. n=6 mice/groups. Data are mean±SE. In theOVA/OVA group serum levels of TARC and eotaxin were increased at 3 and 6h compared to Alum/PBS group; serum levels of TARC were still elevatedat 24 h compared to Alum/PBS group, although to a lesser extent than at3 and 6 h; serum levels of C10 were increased at 3, 6 and 24 h comparedto Alum/PBS group, with maximal induction at 24 h (results shown in FIG.23, FIG. 24, and FIG. 25).

Next, the ability of prophylactic administration of mAb 623 to inhibitserum levels of TARC, eotaxin and C10 using IL-13 humanized mice wasassessed. As will be appreciated by one of skill in the art, this can beused for any of the present antibodies.

Mice were immunized according to the OVA-induced asthma protocoldescribed in reference to FIG. 23, FIG. 24, and FIG. 25. mAb 623 wasadministered intraperitoneally at the dose of 100 μg/mouse (5 mg/kg) ondays 13, 15 and 17 of the study. Control mice received PBS or anirrelevant IgG2 as isotype control. Blood was collected either at 1 and4 hours or 2 and 6 hours after the final challenge. Serum TARC levelswere measured by ELISA (Duoset Kit, R&D System). n=15 mice/group in theOVA group (n=8 for the 1 and 4 hour group and n=7 for the 2 and 6 hourgroup); n=14 mice/group in the OVA+IgG2 group (n=7 for the 1 and 4 hourgroup and n=7 for the 2 and 6 hour group); n=17 mice/group in theOVA+623 group (n=8 for the 1 and 4 hour group and n=9 for the 2 and 6hour group); n=15 mice/group in the Alum group (n=7 for the 1 and 4 hourgroup and n=8 for the 2 and 6 hour group); Data are mean±SE.mAb. mAb 623inhibited the induction of TARC, with a maximum inhibition at the 2 and4 hours time points (see FIG. 26). Thus TARC appears to be an adequatemarker.

To follow the influence on eotaxin, mice were treated as described asdiscussed above in reference to FIG. 26. Serum eotaxin levels weremeasured by ELISA (Duoset Kit, R&D System). Data are mean±SE. mAb 623inhibited eotaxin induction at 1, 2, 4 and 6 hours (see FIG. 27). Thus,eotaxin appears to be an adequate marker.

To follow the influence on C10 levels, mice were treated as describedabove, in reference to FIG. 26. Serum C10 levels were measured by ELISAas described in reference to FIG. 25. Data are mean±SE. mAb 623 hadlittle visible effect on C10 levels at any of the time points tested(FIG. 28). It is believed that, as C10 has a longer time to peak (e.g.,as shown in FIG. 25) and that additional time is likely required to seethe impact on C10 from the antibody as a biomarker and for the use ofC10 as a biomarker.

Thus, by the above disclosed methods, one of skill in the art willreadily be able to identify additional markers that can be of use infollowing IL-13 related disorders and the treatment thereof.

Example 20 Use of Biomarkers in a Subject Receiving MAB 623 and/or MAB731

This example outlines how a biomarker, such as one of the onescharacterized above, can be used to monitor and adjust the amount orfrequency of an antibody that is administered to the subject.

A subject with asthma is identified. The subject is administered astarting amount of mAb 623 and/or 731, e.g., 1-10 mg/kg, everyparticular time unit. Following this, the eotaxin levels in the subjectare measured each hour via ELISA. The amount and/or frequency of the mAbadministered is increased until the level of eotaxin is reduced to alevel indicative of adequate treatment for the subject. This level canbe any significant decrease, for example, any decrease shown in theabove experiments or the maximal decrease achievable through theadministration of the antibody. Once this decrease is observed theamount and/or frequency of administration of the antibody can be heldconstant and even decreased, if appropriate. Thus, biomarkers can allowone to determine the optimal amount of antibody needed.

As will be appreciated by one of skill in the art, this example canallow one of skill in the art to determine what a sufficient ortherapeutically sufficient amount of the antibody can be for eachsubject. By administering a starting amount of an antibody, one canincrease the amount of the antibody, until the presence of the biomarkerbegins to decline, thereby identifying a therapeutically sufficientamount or dose for the subject. Additionally, this can also be used toidentify how (e.g., subcutaneously, intravenously, etc.) and howfrequently (e.g., 1, more than once, a dose per unit of time,continuously, etc.) the antibody should be administered for the desiredresult. As will be appreciated by one of skill in the art, any of theantibodies could be used on any IL-13 related disorder, especially thoselisted herein.

Example 21 The Use of Biomarkers as a Diagnostic

This example details how the biomarkers can be used to identify apatient with an IL-13 related disorder. The level of eotaxin, TARC,and/or C10 present in a healthy individual (e.g., subject without anIL-13 related disorder) is identified. Following this, the level ofeotaxin, TARC, and/or C10′ in other subjects is characterized. Thosewith elevated levels (compared to the healthy individuals) of eotaxin,TARC, and/or C10 will be those that can be suffering from an IL-13related disorder. For further confirmation, subjects with elevatedlevels of eotaxin, TARC, and/or C10 can further have their levels of theother biomarkers compared to the levels of the other biomarker in ahealthy subject.

Thus, this example provides one method that allows one of skill in theart to identify a patient with an IL-13 related disorder or disease.Alternatively, the level of IL-13 in a subject or the properties ofIL-13 in the subject can be examined and compared to those without anIL-13 related disorder. For example, one of the presently disclosedantibodies can be used as a diagnostic, allowing for one to detect anychanges in a subject's available IL-13 and either comparing the detectedamount to a health standard control amount, or for a pre illness amountfor the individual (e.g., an internal control).

As will be appreciated by one of skill in the art, in some embodiments,every IL-13 related disorders will not necessarily have to be associatedwith an increase in eotaxin, TARC or C10. Different diseases might havedifferent biomarkers, some subset of the above, and some IL-13 relateddisorder might not have biomarkers at all.

The above example can be used to help identify an individual sufferingfrom one of the IL-13 related disorders discussed herein. Thus, in someembodiments, the use of the biomarker detectors (e.g., antibodies to thebiomarkers) is as a diagnostic. In other embodiments, such as thatdescribed in Example 5, the antibodies themselves can be used as adiagnostic tool to identify patients with elevated levels of IL-13. Asthe K_(D) of the antibodies is known, one can determine the amount ofIL-13 in a sample via binding of the IL-13 in a sample to the mAb.

Example 22 Determination of IL-13 Related Disorders

This example demonstrates one method by which one can determine whethera disorder is an IL-13 related or dependent disorder. A subject with acandidate disorder is identified. This can be done by randomly selectinga subject from a population. In the alternative, it can be done byselecting a candidate that is demonstrating symptoms that arecharacteristic of one of the disorders disclosed herein. The subject'seotaxin, TARC, and/or C10 levels are examined. The patient isadministered 10 mg/kg of mAb 623 or 731. The subject's eotaxin, TARC,and/or C10 levels are again examined. This can be repeated numeroustimes. If a decrease in the level of the biomarkers is observed, thenthe disorder can be characterized as an IL-13 related disorder.

In alternative embodiments, a greater amount of the antibody is used inprogressive administrations of the antibody. In alternative embodiments,all three biomarkers are examined and all three must show decreases forthe disorder to be characterized as an IL-13 related disorder. Inalternative embodiments, one further correlates a decrease in thesymptoms of the candidate disorder along with the decrease in thebiomarker(s).

As will be appreciated by one of skill in the art, while the presentapplication has outlined numerous antibodies and tested theirfunctionality, one of skill in the art could readily adjust theantibodies by a single amino acid (or several) and obtain a “new”antibody. In order to test such antibody variants, one can repeat any orall of the above disclosed examples to determine if the antibodyfunctions as desired.

As will be appreciated by one of skill in the art, the above examplesoutline how to achieve certain results with particular antibodies;however, in light of the present teaching, one of skill in the art willreadily be able to apply the other antibodies, or other similarantibodies in the above examples and embodiments.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and Examples detail certain preferred embodiments of theinvention and describes the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1. An isolated antibody that specifically binds to IL-13, wherein saidisolated antibody comprises both a light chain variable domain and aheavy chain variable domain and wherein said antibody comprises a) thelight chain of SEQ ID NO:40, or b) the heavy chain of SEQ ID NO:38, orc) the light chain of SEQ ID NO:40 and the heavy chain of SEQ ID NO:38,or d) the light chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:40 asshown in Table 20 and the heavy chain CDR1, CDR2, and CDR3 sequences ofSEQ ID NO:38 as shown in Table 18, or e) the light chain of SEQ IDNO:52, or f) the heavy chain of SEQ ID NO:50, or g) the light chain ofSEQ ID NO:52 and the heavy chain of SEQ ID NO: 50, or h) the light chainCDR1, CDR2, and CDR3 sequences of SEQ ID NO:52 as shown in Table 20 andthe heavy chain CDR1, CDR2, and CDR3 sequences of SEQ ID NO:50 as shownin Table
 18. 2. The isolated antibody of claim 1, wherein said antibodyis a) a human antibody, b) a humanized antibody, c) a chimeric antibody,d) a monoclonal antibody, e) a polyclonal antibody, f) a recombinantantibody, g) an antigen-binding antibody fragment, h) an IgD antibody,i) an IgE antibody, j) an IgG1 antibody, k) an IgG2 antibody, l) an IgG3antibody, or m) an IgG4 antibody.
 3. A composition comprising saidisolated antibody of claim 1 in a pharmaceutically acceptable carrier.